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Abstract:

The present invention provides modified platelets having a reduced
platelet clearance and methods for reducing platelet clearance. Also
provided are compositions for the preservation of platelets. The
invention also provides methods for making a pharmaceutical composition
containing the modified platelets and for administering the
pharmaceutical composition to a mammal to mediate hemostasis.

Claims:

1. A method for treating a platelet using an apparatus, wherein the
apparatus comprises a sterile first container having one or more ports
and containing a preparation of blood cells comprising platelets; and a
second sterile container having one or more ports and containing blood
cell modifying agents; wherein the first container is adapted to the
second container through a sterile conduit reversibly attached to the
first container port and the second container port and the conduit
further comprise a valve; the steps of the method comprise: processing
the platelets using the apparatus such that the platelets are exposed to
the blood cell modifying agents that include UDP-galactose and sialic
acid; wherein the platelets are rendered cold-storage competent after
contacted with the blood modifying agent.

2. The method of claim 1, further comprising separating the leukocytes
from the blood cells prior to exposing the blood cells to the blood cell
modifying agents.

3. The method of claim 1, wherein the blood cells are contacted with the
blood cell modifying agents before infusion of the treated blood cells
into a patient.

4. The method of claim 1, wherein the blood cells are contacted with the
blood cell modifying agents before cold storage of the blood cells.

5. The method of claim 1, wherein the blood cells are contacted with the
blood cell modifying agents at the time of blood collection from a blood
donor.

6. The method of claim 1, further including separating the blood cells
into subpopulations of platelets, plasma, red blood cells and white blood
cells.

7. The method of claim 1, wherein the blood cells are contacted with the
blood cell modifying agents after the blood cells have been separated by
apheresis.

8. A method for treating a platelet using an apparatus, wherein the
apparatus comprises a sterile first container having one or more ports
and containing a preparation of blood cells comprising platelets; and a
second sterile container having one or more ports and containing two or
more blood cell modifying agents; wherein the first container is adapted
to the second container through a sterile conduit reversibly attached to
the first container port and the second container port; the steps of the
method comprise: contacting the platelets with the two or more blood cell
modifying agents, wherein the blood cell modifying agents comprise
UDP-galactose and sialic acid to thereby obtain treated platelets.

9. The method of claim 8, wherein the treated platelets are contacted
with the blood modifying agents in an amount between about 1 micromolar
and about 10 millimolar.

10. The method of claim 8, wherein the treated platelets are contacted
with the blood modifying agents in an amount between about 1 micromolar
and about 1200 micromolar.

11. The method of claim 8, further comprising storing the treated
platelets for a period of time ranging between about 24 hours and about
20 days.

12. The method of claim 8, further comprising storing the treated
platelets for a period of time ranging between about 24 hours and about 7
days.

13. The method of claim 8, further comprising storing the treated
platelets for a period of time ranging between about 24 hours and about 5
days.

14. The method of claim 8, further comprising storing the treated
platelets in a temperature ranging between about 0.degree. C. and about
4.degree. C.

15. The method of claim 8, further comprising storing the treated
platelets in a temperature ranging between about 4.degree. C. and about
15.degree. C.

16. The method of claim 8, further comprising storing the treated
platelets in a temperature ranging between about 15.degree. C. and about
37.degree. C.

17. The method of claim 8, wherein the platelets are contacted with the
UDP-galactose and sialic acid simultaneously or sequentially.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 11/574,857, filed Mar. 7, 2007 (371 date Dec. 31, 2007), which is the
U.S. National stage of International Application No. PCT/US2005/031921,
filed on Sep. 7, 2005, which claims the benefit of U.S. Provisional
Application No. 60/678,724, filed May 6, 2005, U.S. Provisional
Application No. 60/619,176, filed on Oct. 15, 2004, and U.S. Provisional
Application No. 60/607,600, filed on Sep. 7, 2004. The entire teachings
of the above applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The inventions relate to compositions and methods for reducing the
clearance of platelets and prolonging the survival of platelets.

BACKGROUND OF THE INVENTION

[0003] Platelets are anucleate bone marrow-derived blood cells that
protect injured mammals from blood loss by adhering to sites of vascular
injury and by promoting the formation of plasma fibrin clots. Humans
depleted of circulating platelets by bone marrow failure suffer from life
threatening spontaneous bleeding, and less severe deficiencies of
platelets contribute to bleeding complications following trauma or
surgery.

[0004] A reduction in the number of circulating platelets to below
˜70,000 per uL reportedly results in a prolongation of a
standardized cutaneous bleeding time test, and the bleeding interval
prolongs, extrapolating to near infinity as the platelet count falls to
zero. Patients with platelet counts of less than 20,000 per uL are
thought to be highly susceptible to spontaneous hemorrhage from mucosal
surfaces, especially when the thrombocytopenia is caused by bone marrow
failure and when the affected patients are ravaged with sepsis or other
insults. The platelet deficiencies associated with bone marrow disorders
such as a plastic anemia, acute and chronic leukemias, metastatic cancer
but especially resulting from cancer treatment with ionizing radiation
and chemotherapy represent a major public health problem.
Thrombocytopenia associated with major surgery, injury and sepsis also
eventuates in administration of significant numbers of platelet
transfusions.

[0005] A major advance in medical care half a century ago was the
development of platelet transfusions to correct such platelet
deficiencies, and over 9 million platelet transfusions took place in the
United States alone in 1999 (Jacobs et al., 2001). Platelets, however,
unlike all other transplantable tissues, do not tolerate refrigeration,
because they disappear rapidly from the circulation of recipients if
subjected to even very short periods of chilling, and the cooling effect
that shortens platelet survival is irreversible (Becker et al., 1973;
Berger et al., 1998).

[0006] The resulting need to keep these cells at room temperature prior to
transfusion has imposed a unique set of costly and complex logistical
requirements for platelet storage. Because platelets are actively
metabolic at room temperature, they require constant agitation in porous
containers to allow for release of evolved CO2 to prevent the toxic
consequences of metabolic acidosis. Room temperature storage conditions
result in macromolecular degradation and reduced hemostatic functions of
platelets, a set of defects known as "the storage lesion" (Chemoff and
Snyder, 1992). But the major problem with room-temperature storage,
leading to its short (5-day) limitation, is the higher risk of bacterial
infection. Bacterial contamination of blood components is currently the
most frequent infectious complication of blood component use, exceeding
by far that of viral agents (Engelfriet et al., 2000). In the USA,
3000-4500 cases yearly of bacterial sepsis occur because of bacterially
contaminated blood components (Yomtovian et al., 1993).

[0007] The mechanism underlying the unique irreversible cold intolerance
of platelets has been a mystery as has its physiological significance.
Circulating platelets are smooth-surfaced discs that convert to complex
shapes as they react to vascular injury. Over 40 years ago investigators
noted that discoid platelets also change shape at refrigeration
temperatures (Zucker and Borrelli, 1954). Subsequent evidence that a
discoid shape was the best predictor of viability for platelets stored at
room temperature (Schlichter and Harker, 1976) led to the conclusion that
the cold-induced shape change per se was responsible for the rapid
clearance of chilled platelets. Presumably irregularly-shaped platelets
deformed by cooling became entrapped in the microcirculation.

[0008] Based on our studies linking signaling to the mechanisms leading to
platelet shape changes induced by ligands (Hartwig et al., 1995), we
predicted that chilling, by inhibiting calcium extrusion, could elevate
calcium levels to a degree consistent with the activation of the protein
gelsolin, which severs actin filaments and caps barbed ends of actin
filaments. We also reasoned that a membrane lipid phase transition at low
temperatures would cluster phosphoinositides. Phosphoinositide clustering
uncaps actin filament barbed ends (Janmey and Stossel, 1989) to create
nucleation sites for filament elongation. We produced experimental
evidence for both mechanisms, documenting gelsolin activation, actin
filament barbed end uncapping, and actin assembly in cooled platelets
(Hoffmeister et al., 2001; Winokur and Hartwig, 1995). Others have
reported spectroscopic changes in chilled platelets consistent with a
membrane phase transition (Tablin et al., 1996). This information
suggested a method for preserving the discoid shape of chilled platelets,
using a cell-permeable calcium chelator to inhibit the calcium rise and
cytochalasin B to prevent barbed end actin assembly. Although addition of
these agents retained platelets in a discoid shape at 4° C.
(Winokur and Hartwig, 1995), such platelets also clear rapidly from the
circulation, as we report here. Therefore, the problem of the rapid
clearance of chilled platelets remains, and methods of increasing
circulation time as well as storage time for platelets are needed.

SUMMARY OF THE INVENTION

[0009] The present invention provides modified platelets having a reduced
platelet clearance and methods for reducing platelet clearance. Also
provided are compositions and methods for the preservation and storage of
platelets, such as mammalian platelets, particularly human platelets. The
invention also provides methods for making a pharmaceutical composition
containing the modified platelets and for administering the
pharmaceutical composition to a mammal to mediate hemostasis.

[0010] It has now been discovered that cooling of human platelets causes
clustering of the von Willebrand factor (vWf) receptor complex α
subunit (GP1bα) complexes on the platelet surface. The clustering
of GP1bα complexes on the platelet surface elicits recognition by
macrophage complement type three receptors (αMβ2, CR3) in
vitro and in vivo. CR3 receptors recognize N-linked sugars with terminal
βGlcNAc on the surface of platelets, which have formed GP1bα
complexes, and phagocytose the platelets, clearing them from the
circulation and resulting in a concomitant loss of hemostatic function.

[0011] Applicants have discovered that treatment of platelets with an
effective amount of a glycan modifying agent such as N-acetylneuraminic
acid (sialic acid), or certain nucleotide-sugar molecules, such as
CMP-sialic acid or UDP-galactose leads to sialylation or glycation of the
exposed βGlcNAc residues on GP1bα. Effective amounts of a
glycan modifying agent range from about 1 micromolar to about 10
millimolar, about 1 micromolar to about 1 millimolar, and most preferably
about 200 micromolar to about 600 micromolar of the glycan modifying
agent. This has the functional effect of reducing platelet clearance,
blocking platelet phagocytosis, increasing platelet circulation time, and
increasing both platelet storage time and tolerance for temperature
changes. Additionally, platelets removed from a mammal may be stored cold
for extended periods, i.e., at 4 degrees C. for 24 hours, 2 days, 3 days,
5 days, 7 days, 12 days or 20 days or more, without significant loss of
hemostatic function following transplantation. Cold storage provides an
advantage that it inhibits the growth of contaminating microorganisms in
the platelet preparation, important as platelets are typically given to
cancer patients and other immunocompromised patients.

[0012] According to one aspect of the invention, methods for increasing
the circulation time of a population of platelets is provided. The method
comprises contacting an isolated population of platelets with at least
one glycan modifying agent in an amount effective to reduce the clearance
of the population of platelets. In some embodiments, the glycan modifying
agent is selected from the group consisting UDP-galactose and
UDP-galactose precursors. In some preferred embodiments, the glycan
modifying agent is UDP-galactose.

[0013] In some embodiments, the method further comprises adding an enzyme
that catalyzes the modification of a glycan moiety on the platelet. One
example of an enzyme that catalyzes the modification of the glycan moiety
is galactosyl transferase, particularly a beta-1-4-galactosyl
transferase. Another example of an enzyme that catalyzes the modification
of a glycan moiety is a sialyl transferase, which adds sialic acid to the
terminal galactose on the glycan moiety of the platelet.

[0014] In one of the preferred embodiments, the glycan modifying agent is
UDP-galactose and the enzyme that catalyzes the modification of the
glycan moiety is galactosyl transferase. In certain aspects, the glycan
modifying agent further includes a second chemical moiety, which is added
to the glycan on the platelet in a directed manner. An example of this
second chemical moiety is polyethylene glycol (PEG), which when coupled
to the glycan modifying agent such as UDP-galactose as UDP-galactose-PEG,
in the presence of an enzyme such as galactosyl transferase, will
catalyze the addition of PEG to the platelet at the terminus of the
glycan moiety. Thus in certain embodiments, the invention provides for
compositions and methods for the targeted addition of compounds to the
sugars and proteins of cells.

[0015] In some embodiments, the method for increasing the circulation time
of a population of platelets further comprises chilling the population of
platelets prior to, concurrently with, or after contacting the platelets
with the at least one glycan modifying agent.

[0016] In some embodiments, the population of platelets retains
substantially normal hemostatic activity.

[0017] In some embodiments, the step of contacting the population of
platelets with at least one glycan modifying agent is performed in a
platelet bag.

[0018] In some embodiments, the circulation time is increased by at least
about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200%, 500%
or more.

[0019] According to another aspect of the invention, a method for
increasing the storage time of platelets is provided. The method
comprises contacting an isolated population of platelets with an amount
of at least one glycan modifying agent effective to reduce the clearance
of the population of platelets, and storing the population of platelets.
Effective amounts of a glycan modifying agent range from about 1
micromolar to about 1200 micromolar, and most preferably about 200
micromolar to about 600 micromolar of the glycan modifying agent. In
certain aspects the platelet preparation is stored at cold temperatures,
i.e., frozen or refrigerated.

[0020] In some embodiments, the glycan modifying agent is selected from
the group consisting of: a sugar, a monosaccharide sugar, a nucleotide
sugar, sialic acid, sialic acid precursors, CMP-sialic acid,
UDP-galactose, and UDP-galactose precursors. In some embodiments, the
glycan modifying agent is preferably UDP-galactose.

[0021] In some embodiments, the method further comprises adding an
effective amount of an enzyme that catalyzes the addition of the glycan
modifying agent to a glycan on the surface of the platelets. In one of
the preferred embodiments, the glycan modifying agent is UDP-galactose
and the enzyme that catalyzes the addition of the glycan modifying agent
to a glycan on the surface of the platelets is galactosyl transferase,
preferably a beta-1-4-galactosyl transferase. In another preferred
embodiment, the glycan modifying agent is CMP-sialic acid and the enzyme
that catalyzes the addition of the glycan modifying agent to a glycan on
the surface of the platelets is sialyl transferase.

[0022] In some embodiments, the method further comprises chilling the
population of platelets prior to, concurrently with, or after contacting
the platelets with the at least one glycan modifying agent.

[0023] In some embodiments, the population of platelets retains
substantially normal hemostatic activity when transplanted in a mammal.
Prior to transplantation the glycan modifying agent is preferably diluted
or reduced to concentrations of about 50 micromolar or less.

[0024] In certain embodiments, the step of contacting the population of
platelets with at least one glycan modifying agent is performed during
collection of whole blood or collection of the platelets. In certain
embodiments, the glycan modifying agent is introduced into a platelet bag
prior to, concurrently with, or after collection of the platelets.

[0025] The platelets are capable of being stored at reduced temperatures,
for example, frozen, or chilled, and can be stored for extended periods
of time, such as at least about 3 days, at least about 5 days, at least
about 7 days, at least about 10 days, at least about 14 days, at least
about 21 days, or at least about 28 days.

[0026] According to another aspect of the invention, a modified platelet
is provided. The modified platelet comprises a plurality of modified
glycan molecules on the surface of the platelet. The modified glycan
molecules include sialic acid additions to the terminal sugar residues,
or galactosylation of the terminal sugar residues.

[0027] In some embodiments, the modified glycan molecules are moieties of
GP1bα molecules. The modified glycan molecules comprise sialic acid
or at least one added sugar molecule. The added sugar may be a natural
sugar or may be a non-natural sugar. Examples of added sugars include but
are not limited to: nucleotide sugars such as UDP-galactose and
UDP-galactose precursors. In one of the preferred embodiments, the added
nucleotide sugar is CMP-sialic acid or UDP-galactose.

[0028] In another aspect, the invention provides a platelet composition
comprising a plurality of modified platelets. In some embodiments, the
platelet composition further comprises a storage medium. In some
embodiments, the platelet composition further comprises a
pharmaceutically acceptable carrier.

[0029] According to yet another aspect of the invention, a method for
making a pharmaceutical composition for administration to a mammal is
provided. The method comprises the steps of:

[0030] (a) contacting a population of platelets contained in a
pharmaceutically-acceptable carrier with at least one glycan modifying
agent to form a treated platelet preparation,

[0031] (b) storing the treated platelet preparation, and

[0032] (c) warming the treated platelet preparation.

[0033] In some embodiments, the step of warming the treated platelet
preparation is performed by warming the platelets to 37° C.

[0034] In some embodiments, the step of contacting a population of
platelets contained in a pharmaceutically-acceptable carrier with at
least one glycan modifying agent comprises contacting the platelets with
at least one glycan modifying agent, alone or in the presence of an
enzyme that catalyzes the modification of a glycan moiety. The glycan
modifying agent is preferably added at concentrations of about 1
micromolar to about 1200 micromolar, and most preferably about 200
micromolar to about 600 micromolar. In some embodiments, the method
further comprises reducing the concentration of, or removing or
neutralizing the glycan modifying agent or the enzyme in the platelet
preparation. Methods of reducing the concentration of, removing or
neutralizing the glycan modifying agent or enzyme include, for example,
washing the platelet preparation or dilution of the platelet preparation.
The glycan modifying agent is preferably diluted to about 50 micromolar
or less prior to transplantation of the platelets into a human subject.

[0035] Examples of glycan modifying agents are listed above. In one of the
preferred embodiments, the glycan modifying agent is CMP-sialic acid or
UDP-galactose. In some embodiments, the method further comprises adding
an exogenous enzyme that catalyzes the addition of the glycan modifying
agent to a glycan moiety, such as a beta-1-4 galactosyl transferase.

[0036] In one of the preferred embodiments, the glycan modifying agent is
UDP-galactose and the enzyme is galactosyl transferase.

[0037] In some embodiments, the population of platelets demonstrate
substantially normal hemostatic activity, preferably after
transplantation into a mammal.

[0038] In certain embodiments, the step of contacting the population of
platelets with at least one glycan modifying agent is performed during
the collection process on whole blood or fractionated blood, such as on
platelets in a platelet bag.

[0039] In some embodiments, the platelet preparation is stored at a
temperature of less than about 15° C., preferably less than
10° C., and more preferably less than 5° C. In some other
embodiments, the platelet preparation is stored at room temperature. In
other embodiments, the platelets are frozen, e.g., 0° C.,
-20° C., or -80° C. or cooler.

[0040] According to yet another aspect of the invention, a method for
mediating hemostasis in a mammal is provided. The method comprises
administering a plurality of modified platelets or a modified platelet
composition to the mammal. The platelets are modified with the glycan
modifying agent prior to administration, such as during collection, prior
to storing, after storage and during warming, or immediately prior to
transplantation.

[0041] According to still yet another aspect of the invention, a storage
composition for preserving platelets is provided. The composition
comprises at least one glycan modifying agent, added to the platelets in
an amount sufficient to modify platelets glycans, thereby increase the
storage time and/or the circulation time of platelets added to the
storage composition by reducing platelet clearance.

[0042] In some embodiments the composition further comprises an enzyme
that catalyzes the modification of a glycan moiety. The enzyme may be
exogenously added. A beta-1-4 galactosyl transferase or a sialyl
transferase, or both, exemplify preferred enzymes for catalyzing the
modification of the glycan moieties on the platelets.

[0043] According to another aspect of the invention, a container for
collecting (and optionally processing) platelets is provided. The
container comprises at least one glycan modifying agent in an amount
sufficient to modify glycans of platelets contained therein. The
container is preferably a platelet bag, or other blood collection device.

[0044] In some embodiments, the container further comprises an enzyme that
catalyzes the modification of a glycan moiety with the glycan modifying
agent, such as a beta-1-4 galactosyl transferase or a sialyl transferase.

[0045] In some embodiments the container further comprises a plurality of
platelets or plasma comprising a plurality of platelets.

[0046] In some embodiments, the glycan modifying agent is present at a
concentration higher than it is found in naturally occurring platelets or
in serum. In certain aspects these concentrations are 1 micromolar to
1200 micromolar, and most preferably about 200 micromolar to about 600
micromolar. In other embodiments, the beta-1-4 galactosyl transferase or
a sialyl transferase is at a concentration higher than it is found in
naturally occurring platelets or in serum, such as concentrations that
would be observed if the enzyme were added exogenously to the platelets.

[0047] According to still yet another aspect of the invention, a device
for collecting and processing platelets is provided. The device
comprises: a container for collecting platelets; at least one satellite
container in fluid communication with said container; and at least one
glycan modifying agent in the satellite container. The container
optionally includes an enzyme such as a beta-1-4 galactosyl transferase
or a sialyl transferase.

[0048] In some embodiments, the glycan modifying agent in the satellite
container is present in sufficient amounts to preserve the platelets in
the container, for example from concentrations of about 1 micromolar to
about 1200 micromolar.

[0049] In some embodiments, the glycan modifying agent in the satellite
container is prevented from flowing into the container by a breakable
seal.

[0050] In other aspects, the invention includes a kit having a sterile
container capable of receiving and containing a population of platelets,
the container substantially closed to the environment, and a sterile
quantity of a glycan modifying agent sufficient to modify a volume of
platelets collected and stored in the container, the kit further includes
suitable packaging materials and instructions for use. Glycan modifying
agents in the kit include CMP-sialic acid, UDP-galactose, or sialic acid.
The container is suitable for cold-storage of platelets.

[0051] The invention also includes, in certain aspects, a method of
modifying a glycoprotein comprising, obtaining a plurality of platelets
having GP1bα molecules, and contacting the platelets with a glycan
modifying agent, wherein the glycan modifying agent galactosylates or
sialylates the terminus of a GP1bα molecule on the platelets.

[0052] The invention further includes a method of modifying a blood
constituent comprising, obtaining a sample of blood having platelets, and
contacting at least the platelets with a glycan modifying agent, wherein
the glycan modifying agent galactosylates or sialylates the terminus of a
GP1bα molecule on the platelets.

[0053] In other aspects, the invention includes a method of reducing
pathogen growth in a blood sample comprising, obtaining a sample of blood
having platelets, contacting at least the platelets with a glycan
modifying agent, wherein the glycan modifying agent galactosylates or
sialylates the terminus of a GP1bα molecule on the platelets, and
storing the blood sample having modified platelets at a temperature of
about 2 degrees C. to about 18 degrees C. for at least three days,
thereby reducing pathogen growth in the blood sample.

[0054] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first container
having one or more ports and containing a preparation of blood cells, a
second sterile container having one or more ports and containing a blood
cell modifying agent, (also referred to as a platelet solution or a
glycan modifying agent) the first container adapted to the second
container through a sterile conduit reversibly attachable to the first
container port and the second container port, the conduit further
comprising a valve, wherein the blood cell modifying agent is introduced
into the first container and the preparation of blood cells therein is
rendered cold storage competent after the blood cells are contacted with
the blood cell modifying agent. In one embodiment, the invention includes
a sterile third container having one or more ports adapted to the first
container through a second sterile conduit reversibly attachable to the
first container port and the third container port, the conduit further
comprising a valve. In another embodiment, the invention includes a
leukocyte filter. In various embodiments, some shown in the figures, the
first container, second container or the third container are blood bags
or a syringe. In other embodiments, the blood cell modifying agent is a
nucleoside sugar such as UDP galactose, or cytidine
5'monophospho-N-acetylneuraminic acid. The blood cells suitable for
modification in the bioprocess include a population of platelets obtained
from individual random donor blood, pooled random donor blood, or single
donor blood. In various other embodiments, the conduit is adapted to an
in-line filter having a median pore diameter small enough to
substantially prevent the flow of bacteria through the in-line filter.
Preferred median pore diameters for the in-line filter are less than
about 1 micron, more preferably less than about 0.50 microns and most
preferably about 0.22 microns. In yet another embodiment, the second
container port has a frangible barrier. In even another embodiment, the
first conduit or the second conduit reversibly attaches to the first
container port, the second container port or the third container port
through a sterile dock.

[0055] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first container
having one or more ports, and an array having a conduit and a plurality
of sterile docks, wherein each of the sterile docks are reversible
adaptable to blood storage containers, the blood storage containers
having a sample of blood cells and further comprising at least one port
for connecting to the sterile docks of the array, wherein the blood cells
are introduced into the sterile first container through the conduit and
are rendered cold storage competent after the blood cells are contacted
with a blood cell modifying agent introduced into the first container. In
some embodiments, the blood cell modifying agent is a sterile nucleoside
sugar such as UDP galactose or a sterile preparation of cytidine
5'monophospho-N-acetylneuraminic acid. In various other embodiments, the
invention provides an in-line filter having a median pore diameter small
enough to substantially prevent the flow of bacteria through the in-line
filter. Preferred median pore diameters for the in-line filter are less
than about 1 micron, more preferably less than about 0.50 microns and
most preferably about 0.22 microns. In one embodiment, the blood cells
further comprise a population of platelets obtained from individual
random donor blood, pooled random donor blood, or single donor blood. In
another embodiment, the array further comprises a leukocyte filter
proximal to the first container. In even another embodiment, the blood
cell modifying agent is contained in the first to container. In another
embodiment, the invention includes a second container having one or more
ports and containing a blood cell modifying agent, the first container
adapted to the second container through a sterile conduit reversibly
attachable to the first container port and the second container port. In
still yet another embodiment, the second container is a syringe. In one
embodiment, the conduit is adapted to an in-line filter having a median
pore diameter small enough to substantially prevent the flow of bacteria
through the in-line filter. In another embodiment median pore diameters
for the in-line filter are less than about 1 micron, more preferably less
than about 0.50 microns and most preferably about 0.22 microns. In
another embodiment, the second container port has a frangible barrier.

[0056] In another aspect, the invention provides an apparatus for
processing a sample of blood cells, including a sterile first container
having one or more ports the first container further comprising a
subcontainer disposed therein, the subcontainer having a port and a
frangible barrier and containing a blood cell modifying agent, and an
array comprising a conduit and a plurality of sterile docks, wherein each
of the sterile docks are reversible adaptable to blood storage
containers, the blood storage containers having a sample of blood cells
and further comprising at least one port for connecting to the sterile
docks of the array, wherein the blood cells are introduced into the
sterile first container through the conduit and are rendered cold storage
competent after the blood cells are contacted with a blood cell modifying
agent introduced into the first container. In one embodiment, the blood
cell modifying agent is a sterile nucleoside sugar such as UDP galactose
or a sterile preparation of cytidine 5'monophospho-N-acetylneuraminic
acid. In another embodiment median pore diameters for the in-line filter
are less than about 1 micron, more preferably less than about 0.50
microns and most preferably about 0.22 microns. In another embodiment,
the second container port has a frangible barrier. In another embodiment,
the blood cells further comprise a population of individual random donor
blood, pooled random donor blood, or single donor blood. In another
embodiment, the array further comprises a leukocyte filter proximal to
the first container.

[0057] In one aspect, the invention provides a method for treating a blood
cell, including obtaining an apparatus as described, obtaining a sample
of blood cells including a subpopulation of platelets, and exposing the
blood cells to the blood cell modifying agent in the apparatus thereby
rendering the subpopulation of platelets cold-storage competent. In one
embodiment, the method includes separating the leukocytes from the blood
cells prior to exposing the blood cells to the blood cell modifying
agent. In one embodiment, the blood cell modifying agent is a sterile
nucleoside sugar such as UDP galactose or a sterile preparation of
cytidine 5'monophospho-N-acetylneuraminic acid. In another embodiment
median pore diameters for the in-line filter are less than about 1
micron, more preferably less than about 0.50 microns and most preferably
about 0.22 microns. In another embodiment, the second container port has
a frangible barrier. In another embodiment, the blood cells further
comprise a population of individual random donor blood, pooled random
donor blood, or single donor blood. In another embodiment, the method
provides that the blood cells are contacted with the blood cell modifying
agent before infusion of the treated blood cells into a patient. In
another embodiment, the method provides that the blood cells are
contacted with the blood cell modifying agent before cold storage of the
blood cells. In another embodiment, the method provides that the blood
cells are contacted with the blood cell modifying agent at the time of
blood collection from a blood donor. In another embodiment, the method
provides for separating the blood cells into subpopulations of platelets,
plasma, red blood cells and white blood cells. In another embodiment, the
blood cells are contacted with the blood cell modifying agent after the
blood cells have been separated by apheresis.

[0058] In another aspect, the invention provides for a treated blood cell
obtained through the methods described. The treated blood cells,
following cold storage, are suitable for transfusion into a patient.
These and other aspects of the invention, as well as various advantages
and utilities, will be more apparent in reference to the following
detailed description of the invention. Each of the limitations of the
invention can encompass various embodiments of the invention. It is
therefore, anticipated that each of the limitations involving any one
element or combination of elements can be included in each aspect of the
invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059]FIG. 1A shows circulation time in mice of room temperature
platelets and of platelets chilled and rewarmed in the presence or
absence of EGTA-AM and Cytochalasin B. The curves depict the survival of
5-chloromethylfluorescein diacetate (CMFDA) labeled, room temperature
(RT) platelets, platelets chilled at ice-bath temperature (Cold) and
rewarmed to room temperature before injection and chilled and rewarmed
platelets treated with EGTA-AM and cytochalasin B (Cold+CytoB/EGTA) to
preserve their discoid shape. Each curve represents the mean±SD of 6
mice. Identical clearance patterns were observed with
111Indium-labeled platelets.

[0060] FIG. 1B shows that chilled platelets aggregate normally in vitro.
Washed, chilled-rewarmed (Cold) or room temperature (RT) wild type
platelets were stimulated by the addition of the indicated agonists at
37° C. and light transmission was recorded on a standard
aggregometer. Aggregation responses of chilled platelets treated with
EGTA-AM and cytochalasin B were identical to untreated chilled platelets.

[0061] FIG. 1C shows that cold induced clearance occurs predominantly in
the liver of mice. The liver is the primary clearance organ of chilled
platelets, containing 60-90% of injected platelets. In contrast, RT
platelets are cleared more slowly in the spleen. 111Indium labeled
platelets were injected into syngeneic mice and tissues were harvested at
0.5, 1 and 24 hours. Data are expressed per gram of tissue. Each bar
depicts the mean values of 4 animals analyzed±SD.

[0063]FIG. 2 shows that chilled platelets circulate normally in
CR3-deficient mice, but not in complement 3 (C3) or vWf deficient mice.
CMFDA-labeled chilled-rewarmed (Cold) and room temperature (RT) wild type
platelets were transfused into six each of syngeneic wild type (WT),
CR3-deficient (A), vWf-deficient (B) and C3-deficient (C) recipient mice
and their survival times determined. Chilled platelets circulate in
CR3-deficient animals with the same kinetics as room-temperature
platelets, but are cleared rapidly from the circulation of C3- or
vWf-deficient mice. Data are mean±SD for 6 mice.

[0066] FIG. 5 shows GP1bα-CR3 interaction mediates phagocytosis of
chilled human platelets in vitro. FIGS. 5A and 5B show a representative
assay result of THP-1 cells incubated with room temperature (RT) (FIG.
5A) or chilled-rewarmed (Cold) platelets (FIG. 5B). CM-Orange-labeled
platelets associated with macrophages shift in orange fluorescence up the
y axis. The mean percentage of the CM-Orange positive native macrophages
incubated with platelets kept at room temperature was normalized to 1.
Chilling of platelets increases this shift from ˜4% to 20%. The
platelets are predominantly ingested, because they do not dual label with
the FITC-conjugated mAb to CD61. FIG. 5C Undifferentiated (open bars)
THP-1 cells express ˜50% less CR3, and ingest half as many
chilled-rewarmed platelets. Differentiation (filled bars) of CR3
expression however, had no significant effect on the uptake of RT
platelets. Treatment of human platelets with the snake venom
metalloprotease, mocarhagin (Moc), which removes the N-terminus of
GP1bα, from the surface of human platelets (inset; control: solid
line, mocarhagin treated platelets: shaded area), reduced phagocytosis of
chilled platelets by ˜98%. Data shown are means±SD of 5
experiments.

[0067] FIG. 6 shows circulating, chilled platelets have hemostatic
function in CR3 deficient mice. Normal in vivo function of room
temperature (RT) platelets transfused into wild type mice (FIGS. 6A and
6B) and of chilled (Cold) platelets transfused into CR3 deficient mice
(FIGS. 6C and 6D), as determined by their equivalent presence in platelet
aggregates emerging from the wound 24 hrs after infusion of autologous
CMFDA labeled platelets. Peripheral blood (FIGS. 6A and 6C) and the blood
emerging from the wound (shed blood, FIGS. 6B and 6D) were analyzed by
whole blood flow cytometry. Platelets were identified by forward light
scatter characteristics and binding of the PE-conjugated anti-GP1bα
mAb (pOp4). The infused platelets (dots) were identified by their CMFDA
fluorescence and the non-infused platelets (contour lines) by their lack
of CMFDA fluorescence. In the peripheral whole blood samples, analysis
regions were plotted around the GP1bα-positive particles to include
95% of the population on the forward scatter axis (region 1) and the 5%
of particles appearing above this forward light scatter threshold were
defined as aggregates (region 2). The percentages refer to the number of
aggregates formed by CMFDA-positive platelets. This shown result is
representative of 4 experiments. FIG. 6E shows ex vivo function of
CM-Orange, room temperature (RT) platelets transfused into wild type mice
and CM-Orange, chilled-rewarmed (Cold) platelets transfused into CR3
deficient mice, as determined by exposure of P-selectin and fibrinogen
binding following thrombin (1 U/ml) activation of blood drawn from the
mice after 24 hours post infusion. CM-Orange labeled platelets have a
circulation half-life time comparable to that of CMFDA labeled platelets
(not shown). Transfused platelets were identified by their CM-Orange
fluorescence (filled bars). Non-transfused (non-labeled) analyzed
platelets are represented as open bars. Results are expressed as the
percentage of cells present in the P-selectin and fibrinogen positive
regions (region 2). Data are mean±SD for 4 mice.

[0076] FIG. 14 shows that platelets containing galactose transferases on
their surface transfer galactose without the addition of external
transferases as judged by WGA binding (FIG. 14A) and in vitro
phagocytosis results for human platelets (FIG. 14B). FIG. 14C shows that
of UDP-galactose with or without Galactose transferase (GalT) on survival
of murine platelets. UDP-galactose with or without GalT was added to
murine platelets before chilling for 30 min at 37° C. The
platelets were chilled for 2 hours in an ice bath and then transfused
(108 platelets/mouse) into mice and their survival determined.

[0101] FIG. 39 shows the effects of refrigeration and galactosylation on
retention of platelet responses to agonists during storage of
concentrates.

[0102] FIG. 40 shows the effect of storage conditions on shape change
(spreading) and clumping of platelets in concentrates.

[0103]FIG. 41 illustrates an embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
Platelets are derived from a variety of blood sources, including
IRDP--Individual Random Donor Platelets, PRDP--Pooled Random Donor
Platelets and SDP--Single Donor Platelets. The container having the
glycan modifying agent, e.g., a solution of UDP-Gal and/or CMP-NeuAc is
sterile docked to the bag containing the platelets. A sterile dock is
also referred to as a sterile connection device (SCD) or a total
containment device (TCD). The sterile dock permits connection of two
pieces of conduit while maintaining sterility of the system. The glycan
modifying agent is mixed with the platelets and then the modified
platelets are transferred to a non-breathable bag. The glycan modifying
agent can be introduced to the platelets at a variety of times, e.g.,
before infusion, before storage, after componentization or directly to
whole blood, or during the platelet apheresis procedure at the time of
donation. Likewise, the glycan modifying solution may be provided in a
variety of forms, such as full strength concentration liquid,
concentrated liquid--diluted before use, dehydrated, freeze dried,
lyophilisized, powder, frozen, viscous fluid, suspension, base and
activator, or reactant and catalyst. In this embodiment, the blood is
passed through a leukocyte filter. Various methods of leukocyte depletion
are known in the art, e.g., glass wool or other affinity separation
methods for removing leukocyte fractions from whole blood, and provide
examples of means for filtering the leukocytes from the rest of the blood
and specifically the platelets.

[0104]FIG. 42 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
This illustration is similar to FIG. 41 but does not include a leukocyte
filter.

[0105]FIG. 43 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The bag containing the platelets is sterile docked to the bag containing
the platelet solution. The glycans modifying solution, also called a
platelet solution, is mixed with the platelets and then transferred to a
non-breathable bag and thru a leukocyte filter.

[0106]FIG. 44 illustrates a variation of FIG. 43, that does not include a
leukocyte filter.

[0107]FIG. 45 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The syringe containing the platelet solution (UDP-Gal and/or CMP-NeuAc)
is sterile docked to the bag containing the platelets. The platelet
solution is mixed with the platelets and then transferred to a
non-breathable bag and thru a leukocyte filter.

[0108]FIG. 46 illustrates a variation of FIG. 45, that does not include a
leukocyte filter.

[0109] FIG. 47 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The bag containing the platelet solution (UDP-Gal and/or CMP-NeuAc) is
connected to the container port using a bag spike thru a 0.22 micron
filter to the bag containing the platelets. The platelet solution is
mixed with the platelets and then transferred to a non-breathable bag and
thru a leukocyte filter. A 0.22 micron filter is illustrated, but larger
pore diameter filters are suitable to provide increased flow rate. Median
pore sizes greater than about 1 micron are not suitable for sterile
filtration. Preferred sizes are less than about 0.75 microns, more
preferably less than about 0.5 microns, and most preferably about 0.22
microns.

[0110]FIG. 48 illustrates a variation of FIG. 47, that does not include a
leukocyte filter.

[0111]FIG. 49 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The bag containing the platelet solution (either single dose or bulk) is
connected using a luer lock thru a 0.22 micron filter to the bag
containing the platelets. The platelet solution is mixed with the
platelets and then transferred to a non-breathable bag and thru a
leukocyte filter.

[0112] FIG. 50 illustrates a variation of FIG. 49, that does not include a
leukocyte filter.

[0113] FIG. 51 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The syringe containing the platelet solution is connected using a luer
lock thru a 0.22 micron filter to the bag containing the platelets. The
platelet solution is mixed with the platelets and then transferred to a
non-breathable bag and thru a leukocyte filter. Also shown, IRDP can be
pooled to form PRDP.

[0114]FIG. 52 illustrates a variation of FIG. 51, that does not include a
leukocyte filter.

[0115]FIG. 53 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The syringe containing the platelet solution is connected using a luer
lock thru a 0.22 micron filter to the bag containing the platelets. The
platelet solution is mixed with the platelets and then transferred to a
non-breathable bag and thru a leukocyte filter. The syringe can be
aseptically refilled from the bulk platelet solution because of the
in-line filtration device.

[0116]FIG. 54 illustrates a variation of FIG. 53, that does not include a
leukocyte filter.

[0117] FIG. 55 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The large non-breathable bag (final storage bag) containing the platelet
solution includes an array comprising long piece of conduit and a
plurality of ports to allow the sterile docking of multiple IRDP bags
sequentially from the distal end of the tube (denoted #8) to the proximal
end (denoted #1) thru a 0.22 micron filter to the bag containing the
platelet solution. The platelet solution is mixed with the pooled
platelets.

[0118] FIG. 56 illustrates a variation of FIG. 55, that does not include a
leukocyte filter.

[0119]FIG. 57 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
Platelet solution delivery to the containment bag is facilitated by an
SCD on the bag.

[0120]FIG. 58 illustrates a variation of FIG. 57, that does not include a
leukocyte filter.

[0121] FIG. 59 illustrates a variation of FIG. 57. The large
non-breathable bag (final storage bag) has the platelet solution, stored
in a syringe, aseptically connected and added thru a 0.22 micron filter.

[0122] FIG. 60 illustrates a variation of FIG. 59, that does not include a
leukocyte filter.

[0123] FIG. 61 illustrates a variation of the invention, wherein a
container having platelet solution is adapted to the container having
blood cells through a conduit attachable via a luer lock connection. The
conduit has a bag spike to puncture a barrier in the container, thereby
permitting withdrawal of the glycans modifying solution.

[0124] FIG. 62 illustrates a variation of FIG. 61, that does not include a
leukocyte filter.

[0125]FIG. 63 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The large non-breathable bag (final storage bag) has the platelet
solution, stored in a bag, connected with a frangible plug that can be
opened to deliver the platelet solution.

[0126] FIG. 64 illustrates a variation of FIG. 63, that does not include a
leukocyte filter.

[0127] FIG. 65 illustrates another embodiment of the invention wherein a
bioprocess for collecting, treating and storing platelets is described.
The large non-breathable bag (final storage bag) includes an integrated
bag of platelet solution having a frangible plug that can be opened to
deliver the platelet solution directly into the platelet storage
container.

[0128] FIG. 66 illustrates a variation of the embodiment illustrated as
FIG. 65. The bag having the platelet modifying solution is integrated
within the storage bag. The platelet solution is released upon breaking
of the frangible plug or separation membrane.

DETAILED DESCRIPTION OF THE INVENTION

[0129] The invention provides a population of modified platelets that have
enhanced circulation properties and that retain substantially normal in
vivo hemostatic activity. Hemostatic activity refers broadly to the
ability of a population of platelets to mediate bleeding cessation.
Various assays are available for determining platelet hemostatic activity
(Bennett, J. S, and Shattil, S. J., 1990, "Platelet function,"
Hematology, Williams, W. J., et al., Eds. McGraw Hill, pp 1233-12250).
However, demonstration of "hemostasis" or "hemostatic activity"
ultimately requires a demonstration that platelets infused into a
thrombocytopenic or thrombopathic (i.e., non-functional platelets) animal
or human circulate and stop natural or experimentally-induced bleeding.

[0130] Short of such a demonstration, laboratories use in vitro tests as
surrogates for determining hemostatic activity. These tests, which
include assays of aggregation, secretion, platelet morphology and
metabolic changes, measure a wide variety of platelet functional
responses to activation. It is generally accepted in the art that the in
vitro tests are reasonably indicative of hemostatic function in vivo.

[0131] Substantially normal hemostatic activity refers to an amount of
hemostatic activity seen in the modified platelets, that is functionally
equivalent to or substantially similar to the hemostatic activity of
untreated platelets in vivo, in a healthy (non-thrombocytopenic or
non-thrombopathic mammal) or functionally equivalent to or substantially
similar to the hemostatic activity of a freshly isolated population of
platelets in vitro.

[0132] The instant invention provides methods for reduced temperature
storage of platelets which increases the storage time of the platelets,
as well as methods for reducing clearance of or increasing circulation
time of a population of platelets in a mammal. Also provided are platelet
compositions methods and compositions for the preservation of platelets
with preserved hemostatic activity as well as methods for making a
pharmaceutical composition containing the preserved platelets and for
administering the pharmaceutical composition to a mammal to mediate
hemostasis. Also provided are kits for treating a platelet preparation
for storage, and containers for storing the same.

[0133] In one aspect of the invention, the method for increasing
circulation time of an isolated population of platelets involves
contacting an isolated population of platelets with at least one glycan
modifying agent in an amount effective to reduce the clearance of the
population of platelets. As used herein, a population of platelets refers
to a sample having one or more platelets. A population of platelets
includes a platelet concentrate. The term "isolated" means separated from
its native environment and present in sufficient quantity to permit its
identification or use. As used herein with respect to a population of
platelets, isolated means removed or cleared from the blood circulation
of a mammal. The circulation time of a population of platelets is defined
as the time when one-half of the platelets in that population are no
longer circulating in a mammal after transplantation into that mammal. As
used herein, "clearance" means removal of the modified platelets from the
blood circulation of a mammal (such as but not limited to by macrophage
phagocytosis). As used herein, clearance of a population of platelets
refers to the removal of a population of platelets from a unit volume of
blood or serum per unit of time. Reducing the clearance of a population
of platelets refers to preventing, delaying, or reducing the clearance of
the population of platelets. Reducing clearance of platelets also may
mean reducing the rate of platelet clearance.

[0134] A glycan modifying agent refers to an agent that modifies glycan
residues on the platelet. As used herein, a "glycan" or "glycan residue"
is a polysaccharide moiety on surface of the platelet, exemplified by the
GP1bα polysaccharide. A "terminal" glycan or glycan residue is the
glycan at the distal terminus of the polysaccharide, which typically is
attached to polypeptides on the platelet surface. Preferably, the glycan
modifying agent alters GP1bα on the surface of the platelet.

[0136] UDP-galactose is an intermediate in galactose metabolism, formed by
the enzyme UDP-glucose-α-D-galactose-1-phosphate
uridylyltransferase which catalyzes the release of glucose-1-phosphate
from UDP-glucose in exchange for galactose-1-phosphate to make
UDP-galactose. UDP-galactose and sialic acid are widely available from
several commercial suppliers such as Sigma. In addition, methods for
synthesis and production of UDP-galactose are well known in the art and
described in the literature (see for example, Liu et al, ChemBioChem 3,
348-355, 2002; Heidlas et al, J. Org. Chem. 57, 152-157; Butler et al,
Nat. Biotechnol. 8, 281-284, 2000; Koizumi et al, Carbohydr. Res. 316,
179-183, 1999; Endo et al, Appl. Microbiol., Biotechnol. 53, 257-261,
2000). UDP-galactose precursors are molecules, compounds, or intermediate
compounds that may be converted (e.g., enzymatically or biochemically) to
UDP-galactose. One non-limiting example of a UDP-galactose precursor is
UDP-glucose. In certain embodiments, an enzyme that converts a
UDP-galactose precursor to UDP-galactose is added to a reaction mixture
(e.g. in a platelet container).

[0137] An effective amount of a glycan modifying agent is that amount of
the glycan modifying agent that alters a sufficient number of glycan
residues on the surface of platelets, that when introduced to a
population of platelets, increases circulation time and/or reduces the
clearance of the population of platelets in a mammal following
transplantation of the platelets into the mammal. An effective amount of
a glycan modifying agent is a concentration from about 1 micromolar to
about 1200 micromolar, preferably from about 10 micromolar to about 1000
micromolar, more preferably from about 100 micromolar to about 750
micromolar, and most preferably from about 200 micromolar to about 600
micromolar.

[0138] Modification of platelets with glycan modifying agents can be
preformed as follows. The population of platelets is incubated with the
selected glycan modifying agent (concentrations of 1-1200 μM) for at
least 1, 2, 5, 10, 20, 40, 60, 120, 180, 240, or 300 min. at 22°
C.-37° C. Multiple glycan modifying agents (i.e., two, three four
or more) may be used simultaneously or sequentially. In some embodiments
0.1-500 mU/ml galactose transferase or sialyl transferase is added to the
population of platelets. Galactose transfer can be monitored functionally
using FITC-WGA (wheat germ agglutinin) binding. The goal of the glycan
modification reaction is to reduce WGA binding to resting room
temperature WGA binding-levels. Galactose transfer can be quantified
using 14C-UDP-galactose. Non-radioactive UDP-galactose is mixed with
14C-UDP-galactose to obtain appropriate galactose transfer.
Platelets are extensively washed, and the incorporated radioactivity
measured using a γ-counter. The measured cpm permits calculation of
the incorporated galactose. Similar techniques are applicable to
monitoring sialic acid transfer.

[0139] Reducing the clearance of a platelet encompasses reducing clearance
of platelets after storage at room temperature, or after chilling, as
well as "cold-induced platelet activation". Cold-induced platelet
activation is a term having a particular meaning to one of ordinary skill
in the art. Cold-induced platelet activation may manifest by changes in
platelet morphology, some of which are similar to the changes that result
following platelet activation by, for example, contact with glass. The
structural changes indicative of cold-induced platelet activation are
most easily identified using techniques such as light or electron
microscopy. On a molecular level, cold-induced platelet activation
results in actin bundle formation and a subsequent increase in the
concentration of intracellular calcium. Actin-bundle formation is
detected using, for example, electron microscopy. An increase in
intracellular calcium concentration is determined, for example, by
employing fluorescent intracellular calcium chelators. Many of the
above-described chelators for inhibiting actin filament severing are also
useful for determining the concentration of intracellular calcium (Tsien,
R., 1980, supra.). Accordingly, various techniques are available to
determine whether or not platelets have experienced cold-induced
activation.

[0140] The effect of galactose or sialic acid addition to the glycan
moieties on platelets, resulting in diminished clearance of modified
platelets, can be measured for example using either an in vitro system
employing differentiated THP-1 cells or murine macrophages, isolated from
the peritoneal cavity after thioglycolate injection stimulation. The rate
of clearance of modified platelets compared to unmodified platelets is
determined. To test clearance rates, the modified platelets are fed to
the macrophages and ingestion of the platelets by the macrophages is
monitored. Reduced ingestion of modified platelets relative to unmodified
platelets (twofold or greater) indicates successful modification of the
glycan moiety for the purposes described herein.

[0141] In accordance with the invention, the population of modified
platelets can be chilled without the deleterious effects (cold-induced
platelet activation) usually experienced on chilling of untreated
platelets. The population of modified platelets can be chilled prior to,
concurrently with, or after contacting the platelets with the at least
one glycan modifying agent. The selective modification of glycan moieties
reduces clearance, following chilling (also if not chilled), thus
permitting longer-term storage than is presently possible. As used
herein, chilling refers to lowering the temperature of the population of
platelets to a temperature that is less than about 37° C. In some
embodiments, the platelets are chilled to a temperature that is less than
about 15° C. In some preferred embodiments, the platelets are
chilled to a temperature ranging from between about 0° C. to about
4° C. Chilling also encompasses freezing the platelet preparation,
i.e., to temperatures less than 0° C., -20° C., -50°
C., and -80° C. or cooler. Process for the cryopreservation of
cells are well known in the art.

[0142] In some embodiments, the population of platelets is stored chilled
for at least 3 days. In some embodiments, the population of platelets is
stored chilled for at least 5, 7, 10, 14, 21, and 28 days or longer.

[0143] In some embodiments of the invention, the circulation time of the
population of platelets is increased by at least about 10%. In some other
embodiments, the circulation time of the population of platelets is
increased by at least about 25%. In yet some other embodiments, the
circulation time of the population of platelets is increased by at least
about 50% to about 100%. In still yet other embodiments, the circulation
time of the population of platelets is increased by about 150% or
greater.

[0144] The invention also embraces a method for increasing the storage
time of platelets. As used herein the storage time of platelets is
defined as the time that platelets can be stored without substantial loss
of platelet function or hemostatic activity such as the loss of the
ability to circulate or increased platelet clearance.

[0145] The platelets are collected from peripheral blood by standard
techniques known to those of ordinary skill in the art, for example by
isolation from whole blood or by apheresis processes. In some
embodiments, the platelets are contained in a pharmaceutically-acceptable
carrier prior to treatment with a glycan modifying agent.

[0146] According to another aspect of the invention, a modified platelet
or a population of modified platelets is provided. The modified platelet
comprises a plurality of modified glycan molecules on the surface of the
platelet. In some embodiments, the modified glycan moieties are
GP1bα, molecules. The invention also encompasses a platelet
composition in a storage medium. In some embodiments the storage medium
comprises a pharmaceutically acceptable carrier.

[0147] The term "pharmaceutically acceptable" means a non-toxic material
that does not interfere with the effectiveness of the biological activity
of the platelets and that is a non-toxic material that is compatible with
a biological system such as a cell, cell culture, tissue, or organism.
Pharmaceutically acceptable carriers include diluents, fillers, salts,
buffers, stabilizers, solubilizers, and other materials which are well
known in the art, for example, a buffer that stabilizes the platelet
preparation to a pH of 7.4, the physiological pH of blood, is a
pharmaceutically acceptable composition suitable for use with the present
invention.

[0148] The invention further embraces a method for making a pharmaceutical
composition for administration to a mammal. The method comprises
preparing the above-described platelet preparation, and warming the
platelet preparation. In some embodiments, the method comprises
neutralizing, removing or diluting the glycan modifying agent(s) and/or
the enzyme(s) that catalyze the modification of the glycan moiety, and
placing the modified platelet preparation in a pharmaceutically
acceptable carrier. In a preferred embodiment, the chilled platelets are
warmed to room temperature (about 22° C.) prior to neutralization
or dilution. In some embodiments, the platelets are contained in a
pharmaceutically acceptable carrier prior to contact with the glycan
modifying agent(s) with or without the enzyme(s) that catalyze the
modification of the glycan moiety and it is not necessary to place the
platelet preparation in a pharmaceutically acceptable carrier following
neutralization or dilution.

[0149] As used herein, the terms "neutralize" or "neutralization" refer to
a process by which the glycan modifying agent(s) and/or the enzyme(s)
that catalyze the modification of the glycan moiety are rendered
substantially incapable of glycan modification of the glycan residues on
the platelets, or their concentration in the platelet solution is lowered
to levels that are not harmful to a mammal, for example, less that 50
micromolar of the glycan modifying agent. In some embodiments, the
chilled platelets are neutralized by dilution, e.g., with a suspension of
red blood cells. Alternatively, the treated platelets can be infused into
the recipient, which is equivalent to dilution into a red blood cell
suspension. This method of neutralization advantageously maintains a
closed system and minimizes damage to the platelets. In a preferred
embodiment of glycan modifying agents, no neutralization is required.

[0150] An alternative method to reduce toxicity is by inserting a filter
in the infusion line, the filter containing, e.g. activated charcoal or
an immobilized antibody, to remove the glycan modifying agent(s) and/or
the enzyme(s) that catalyze the modification of the glycan moiety.

[0151] Either or both of the glycan modifying agent(s) and the enzyme(s)
that catalyze the modification of the glycan moiety also may be removed
or substantially diluted by washing the modified platelets in accordance
with standard clinical cell washing techniques.

[0152] The invention further provides a method for mediating hemostasis in
a mammal. The method includes administering the above-described
pharmaceutical preparation to the mammal. Administration of the modified
platelets may be in accordance with standard methods known in the art.
According to one embodiment, a human patient is transfused with red blood
cells before, after or during administration of the modified platelets.
The red blood cell transfusion serves to dilute the administered,
modified platelets, thereby neutralizing the glycan modifying agent(s)
and the enzyme(s) that catalyze the modification of the glycan moiety.

[0153] The dosage regimen for mediating hemostasis using the modified
platelets is selected in accordance with a variety of factors, including
the type, age, weight, sex and medical condition of the subject, the
severity of the disease, the route and frequency of administration. An
ordinarily skilled physician or clinician can readily determine and
prescribe the effective amount of modified platelets required to mediate
hemostasis.

[0154] The dosage regimen can be determined, for example, by following the
response to the treatment in terms clinical signs and laboratory tests.
Examples of such clinical signs and laboratory tests are well known in
the art and are described, see, Harrison's Principles of Internal
Medicine, 15th Ed., Fauci A S et al., eds., McGraw-Hill, New York, 2001.

[0155] Also within the scope of the invention are storage compositions and
pharmaceutical compositions for mediating hemostasis. In one embodiment,
the compositions comprise a pharmaceutically-acceptable carrier, a
plurality of modified platelets, a plurality of glycan modifying agent(s)
and optionally the enzyme(s) that catalyze the modification of the glycan
moiety. The glycan modifying agent(s) and the enzyme(s) that catalyze the
modification of the glycan moiety are present in the composition in
sufficient amounts so as to reduce platelet clearance. Preferably, glycan
modifying agent(s) (and optionally the enzyme(s) that catalyze the
modification of the glycan moiety) are present in amounts whereby after
chilling and neutralization, the platelets maintain substantially normal
hemostatic activity. The amounts of glycan modifying agent(s) (and
optionally the enzyme(s) that catalyze the modification of the glycan
moiety) which reduce platelet clearance can be selected by exposing a
preparation of platelets to increasing amounts of these agents, exposing
the treated platelets to a chilling temperature and determining (e.g., by
microscopy) whether or not cold-induced platelet activation has occurred.
Preferably, the amounts of glycan modifying agent(s) and the enzyme(s)
that catalyze the modification of the glycan moiety can be determined
functionally by exposing the platelets to varying amounts of glycan
modifying agent(s) and the enzyme(s) that catalyze the modification of
the glycan moiety, chilling the platelets as described herein, warming
the treated (chilled) platelets, optionally neutralizing the platelets
and testing the platelets in a hemostatic activity assay to determine
whether the treated platelets have maintained substantially normal
hemostatic activity.

[0156] For example, to determine the optimal concentrations and conditions
for preventing cold-induced activation of platelets by modifying them
with a glycan modifying agent(s) (and optionally the enzyme(s) that
catalyze the modification of the glycan moiety), increasing amounts of
these agents are contacted with the platelets prior to exposing the
platelets to a chilling temperature. The optimal concentrations of the
glycan modifying agent(s) and the enzyme(s) that catalyze the
modification of the glycan moiety are the minimal effective
concentrations that preserve intact platelet function as determined by in
vitro tests (e.g., observing morphological changes in response to glass,
thrombin, cryopreservation temperatures; ADP-induced aggregation)
followed by in vivo tests indicative of hemostatic function (e.g.,
recovery, survival and shortening of bleeding time in a thrombocytopenic
animal or recovery and survival of 51Cr-labeled platelets in human
subjects).

[0157] According to yet another aspect of the invention, a composition for
addition to platelets to reduce platelet clearance or to increase
platelet storage time is provided. The composition includes one or more
glycan modifying agents. In certain embodiments, the composition also
includes an enzyme(s) that catalyze the modification of the glycan
moiety. The glycan modifying agent and the enzyme(s) that catalyzes the
modification of the glycan moiety are present in the composition in
amounts that prevent cold-induced platelet activation.

[0158] The invention also embraces a storage composition for preserving
platelets. The storage composition comprises at least one glycan
modifying agent in an amount sufficient to reduce platelet clearance. In
some embodiments the storage composition further comprises an enzyme that
catalyzes the modification of a glycan moiety on the platelet. The glycan
modifying agent is added to the population of platelets that are
preferably kept between about room temperature and 37° C. In some
embodiments, following treatment, the population of platelets is cooled
to about 4° C. In some embodiments, the platelets are collected
into a platelet pack, bag, or container according to standard methods
known to one of skill in the art. Typically, blood from a donor is drawn
into a primary container which may be joined to at least one satellite
container, all of which containers are connected and sterilized before
use. In some embodiments, the satellite container is connected to the
container for collecting platelets by a breakable seal. In some
embodiments, the primary container further comprises plasma containing a
plurality of platelets.

[0159] In some embodiments, the platelets are concentrated (e.g. by
centrifugation) and the plasma and red blood cells are drawn off into
separate satellite bags (to avoid modification of these clinically
valuable fractions) prior to adding the glycan modifying agent with or
without the enzyme that catalyzes the modification of a glycan moiety on
the platelet. Platelet concentration prior to treatment also may minimize
the amounts of glycan modifying agents required for reducing the platelet
clearance, thereby minimizing the amounts of these agents that are
eventually infused into the patient.

[0160] In one embodiment, the glycan modifying agent(s) are contacted with
the platelets in a closed system, e.g. a sterile, sealed platelet pack,
so as to avoid microbial contamination. Typically, a venipuncture conduit
is the only opening in the pack during platelet procurement or
transfusion. Accordingly, to maintain a closed system during treatment of
the platelets with the glycan modifying agent(s), the agent(s) is placed
in a relatively small, sterile container which is attached to the
platelet pack by a sterile connection tube (see e.g., U.S. Pat. No.
4,412,835, the contents of which are incorporated herein by reference).
The connection tube may be reversibly sealed, or have a breakable seal,
as will be known to those of skill in the art. After the platelets are
concentrated, e.g. by allowing the platelets to settle and squeezing the
plasma out of the primary pack and into a second bag according to
standard practice, the seal to the container(s) including the glycan
modifying agent(s) is opened and the agents are introduced into the
platelet pack. In one embodiment, the glycan modifying agents are
contained in separate containers having separate resealable connection
tubes to permit the sequential addition of the glycan modifying agents to
the platelet concentrate.

[0161] Following contact with the glycan modifying agent(s), the treated
platelets are chilled. In contrast to platelets stored at, for example,
22° C., platelets stored at cryopreservation temperatures have
substantially reduced metabolic activity. Thus, platelets stored at
4° C. are metabolically less active and therefore do not generate
large amounts of CO2 compared with platelets stored at, for example,
22° C. (Slichter, S. J., 1981; Vox Sang 40 (Suppl 1), pp 72-86,
Clinical Testing and Laboratory-Clinical correlations). Dissolution of
CO2 in the platelet matrix results in a reduction in pH and a
concomitant reduction in platelet viability (Slichter, S., 1981, supra.).
This can be resolved by adding buffers to the platelet population, the
buffers selected to keep the platelet population at or near the
physiological pH of blood. Likewise, conventional platelet packs are
formed of materials that are designed and constructed of a sufficiently
permeable material to maximize gas transport into and out of the pack
(O2 in and CO2 out). The prior art limitations in platelet pack
design and construction are obviated by the instant invention, which
permits storage of platelets at cryopreservation temperatures, thereby
substantially reducing platelet metabolism and diminishing the amount of
CO2 generated by the platelets during storage. Accordingly, the
invention further provides platelet containers that are substantially
non-permeable to CO2 and/or O2, which containers are useful
particularly for cold storage of platelets. In both the gas permeable and
non-gas permeable embodiments, the invention provides for a blood storage
container having therein, a quantity of a glycan modifying agent
sufficient to substantially modify the carbohydrates of the platelets
introduced therein, such that the platelets become capable of cold
storage and subsequent in vivo circulation.

[0162] The present invention also provides for kits that are used for
platelet collection, processing and storage, further including suitable
packaging materials and instructions for using the kit contents. It is
preferred that all reagents and supplies in the kit be sterile, in
accordance with standard medical practices involving the handling and
storage of blood and blood products. Methods for sterilizing the kit
contents are known in the art, for example, ethylene gas, irradiation and
the like. In certain embodiments, the kit may include venipuncture
supplies and/or blood collection supplies, for example a needle set,
solution for sterilizing the skin of a platelet donor, and a blood
collection bag or container. Preferably the container is "closed", i.e.,
substantially sealed from the environment. Such closed blood collection
containers are well known in the art, and provide a means of preventing
microbial contamination of the platelet preparation contained therein.
Other embodiments include kits containing supplies for blood collection
and platelet apheresis. The kits may further include a quantity of the
glycan modifying agent, sufficient to modify the volume of platelets
collected and stored in the container. In certain embodiments, the kit
includes reagents for modifying the terminal glycan of platelets with a
second or third chemical moiety, for example to PEGylate collected
platelets. In other embodiments, the kit includes a blood collection
system having a blood storage container wherein the glycan modifying
agent is provided within the container in an amount sufficient to treat
the volume of blood or platelets held by the container. The quantity of
glycan modifying agent will depend on the volume of the container. It is
preferred the glycan modifying agent be provided as a sterile
non-pyogenic solution, but it may also be supplied as a lyophilized
powder. For example, a blood bag is provided having a capacity of 250 ml.
Contained in the blood bag is a quantity of UDP-Gal such that when 250 ml
of blood is added, the final concentration of the UDP-Gal is
approximately 200 micromolar. Other embodiments contain different
concentrations of glycan modifying agents, for example but not limited to
quantities resulting in final concentrations of 10 micromolar to 10
millimolar, and preferably 100 micromolar to 1 millimolar of the glycan
modifying agents. Other embodiments use combinations of glycan modifying
agents, e.g., to effect sialylation or galactosylation of N-linked
glycoproteins on blood products introduced into the container.

[0163] The invention will be more fully understood by reference to the
following examples. These examples, however, are merely intended to
illustrate the embodiments of the invention and are not to be construed
to limit the scope of the invention.

EXAMPLES

Example 1

Introduction

[0164] Modest cooling primes platelets for activation, but refrigeration
causes shape changes and rapid clearance, compromising storage of
platelets for therapeutic transfusions. We found that shape change
inhibition does not normalize cold-induced clearance. We also found that
cooling platelets rearranges the surface configuration of the von
Willebrand factor (vWf) receptor complex α subunit (GP1bα)
such that it becomes targeted for recognition by complement receptor 3
receptors (CR3) predominantly expressed on liver macrophages, leading to
platelet phagocytosis and clearance. GP1b α removal prolongs
survival of unchilled platelets. Chilled platelets bind vWf and function
normally in vitro and ex vivo after transfusion into CR3-deficient mice.
Cooled platelets, however, are not "activated" like platelets exposed to
thrombin or ADP, and their vWf-receptor complex reacts normally with
activated vWf.

[0165] As the temperature falls below 37° C. platelets become more
susceptible to activation by thrombotic stimuli, a phenomenon known as
"priming" (Faraday and Rosenfeld, 1998; Hoffmeister et al., 2001).
Priming may be an adaptation to limit bleeding at lower temperatures of
body surfaces where most injuries occur. We propose that the hepatic
clearance system's purpose is to remove repeatedly primed platelets, and
that conformational changes in GP1bα that promote this clearance do
not affect GP1bα's hemostatically important binding to vWf.
Therefore, selective modification of GP1bα may accommodate cold
storage of platelets for transfusion.

[0169] The N-terminus of GP1bα was enzymatically removed from the
surface of chilled or room temperature maintained and labeled platelets
in buffer B, also containing 1 mM Ca2+ and 10 μg/ml of the snake
venom metalloprotease mocarhagin (Ward et al., 1996). After the enzymatic
digestion, the platelets were washed by centrifugation with 5×
volume of buffer A and routinely checked by microscopy for aggregates.
GP1bα-N-terminus removal was monitored by incubating platelet
suspensions with 5 μg/ml of FITC-conjugated anti-human GP1bα
(SZ2) mAb for 10 min at room temperature and followed by immediate flow
cytometry analysis on a FACScalibur Flow Cytometer (Becton Dickinson
Biosciences, San Jose, Calif.). Platelets were gated by forward/side
scatter characteristics and 50,000 events acquired.

Murine Platelets

[0170] Mice were anesthetized with 3.75 mg/g (2.5%) of Avertin, and 1 ml
blood was obtained from the retroorbital eye plexus into 0.1 volume of
Aster-Jandl anticoagulant. PRP was prepared by centrifugation of
anticoagulated blood at 300×g for 8 min at room temperature.
Platelets were separated from plasma proteins by centrifugation at
1200×g for 5 min and washed two times by centrifugation
(1200×g for 5 min) using 5× volumes of washing buffer (buffer
A). This procedure is meant by subsequent use of the term "washed".
Platelets were resuspended at a concentration of 1×109/ml in a
solution containing 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl2, 5 mM
NaHCO3, 10 mM glucose and 10 mM Hepes, pH 7.4 (buffer B). Platelet
count was determined using a Bright Line Hemocytometer (Hausser
Scientific, Horsham, Pa.) under a phase-contrast microscope at 400×
magnification. Some radioactive platelet clearance studies were performed
with 111Indium, and we labeled mouse platelets using a method
described for primate platelets (Kotze et al., 1985). Platelets were
resuspended at a concentration of 2×109/ml in 0.9% NaCl, pH
6.5 (adjusted with 0.1 M sodium citrate), followed by the addition of 500
μCi 111Indium chloride for 30 min at 37° C. and washed as
described above and suspended in buffer B at a concentration of
1×109/ml.

[0171] For intravital microscopy or other platelet survival experiments,
washed platelets were labeled either with 2.5 μM CellTracker Green
CMFDA (5-chloromethyl fluorescein diacetate) (CMFDA) for 20 min at
37° C. (Baker et al., 1997) or with 0.15 μM TRITC for 20 min at
37° C. in buffer B also containing 0.001% DMSO, 20 mM HEPES.
Unincorporated dye was removed by centrifugation as described above, and
platelets were suspended at a concentration of 1×109/ml in
buffer B.

[0172] The N-terminus of GP1bα was enzymatically removed from the
surface of chilled or room temperature labeled platelets with 100
μg/ml O-sialoglycoprotein endopeptidase in buffer B containing 1 mM
Ca2+ for 20 min at 37° C. (Bergmeier et al., 2001). After
enzymatic digestion, platelets were washed by centrifugation and checked
by light microscopy for aggregates. Enzymatic removal of the
GP1bα-N-terminus removal was monitored by incubating the platelet
suspensions with 5 μg/ml of PE-conjugated anti-mouse GP1bα mAb
pOp4 for 10 min at room temperature, and bound PE analyzed by flow
cytometry.

[0173] To inhibit cold-induced platelet shape changes, 109/ml
platelets in buffer B were loaded with 2 μM EGTA-AM followed by 2
μM cytochalasin B as previously described (Winokur and Hartwig, 1995),
labeled with 2.5 μM CMFDA for 30 min at 37° C. and then chilled
or maintained at room temperature. The platelets were subjected to
standard washing and suspended at a concentration of 1×109/ml
in buffer B before injection into mice.

Platelet Temperature Protocols

[0174] To study the effects of temperature on platelet survival or
function, unlabeled, radioactively labeled, or fluorescently-labeled
mouse or human platelets were incubated for 2 hours at room temperature
(25-27° C.) or else at ice bath temperatures and then rewarmed for
15 minutes at 37° C. before transfusion into mice or in vitro
analysis. Platelets subjected to these treatments are designated cooled
or chilled (or chilled, rewarmed) and room temperature platelets
respectively.

Murine Platelet Recovery, Survival and Fate

[0175] CMFDA labeled chilled or room temperature murine platelets
(108) were injected into syngeneic mice via the lateral tail vein
using a 27-gauge needle. For recovery and survival determination, blood
samples were collected immediately (<2 min) and 0.5, 2, 24, 48, 72
hours after transfusion into 0.1 volume of Aster-Jandl anticoagulant.
Whole blood analysis using flow cytometry was performed and the
percentage of CMFDA positive platelets determined by gating on all
platelets according to their forward and side scatter characteristics
(Baker et al., 1997). 50,000 events were collected in each sample. CMFDA
positive platelets measured at a time <2 min was set as 100%. The
input of transfused platelets per mouse was ˜2.5-3% of the whole
platelet population.

[0176] To evaluate the fate of platelets, tissues (heart, lung, liver,
spleen, muscle, and femur) were harvested at 0.5, 1 and 24 hours after
the injection of 108 chilled or room temperature 111Indium
labeled platelets into mice. The organ-weight and their radioactivity
were determined using a Wallac 1470 Wizard automatic gamma counter
(Wallac Inc., Gaithersburg, Md.). The data were expressed as gamma count
per gram organ. For recovery and survival determination of radioactive
platelets, blood samples were collected immediately (<2 min) and 0.5
and hours after transfusion into 0.1 volume of Aster-Jandl anticoagulant
and their gamma counts determined (Kotze et al., 1985).

Platelet Aggregation

[0177] Conventional tests were performed and monitored in a Bio/Data
aggregometer (Horsham, Pa.). Samples of 0.3-ml murine washed and stirred
platelets were exposed to 1 U/ml thrombin, 10 μM ADP, or 3 μg/ml
CRP at 37° C. Light transmission was recorded over 3 min.

[0179] Resting mouse platelets maintained at room temperature or chilled 2
hrs were diluted to a concentration of 2×106/ml in phosphate
buffered saline (PBS) containing 0.05% glutaraldehyde. Platelet solutions
(200 μl) were placed on a polylysine-coated glass coverslip contained
in wells of 96-well plate, and the platelets were adhered to each
coverslip by centrifugation at 1,500×.g for 5 min at room
temperature. The supernatant fluid was then removed, and platelets bound
to the coverslip were fixed with 0.5% glutaraldehyde in PBS for 10 min.
The fixative was removed, unreacted aldehydes quenched with a solution
containing 0.1% sodium borohydride in PBS followed by washing with PBS
containing 10% BSA. GP1bα on the platelet surface was labeled with
a mixture of three rat anti-mouse GP1bα monoclonal antibodies, each
at 10 μg/ml (Bergmeier et al., 2000) for 1 hr followed by 10 nm gold
coated with goat anti-rat IgG. The coverslips were extensively washed
with PBS, post-fixed with 1% glutaraldehyde, washed again with distilled
water, rapidly frozen, freeze-dried, and rotary coated with 1.2 nm of
platinum followed by 4 nm of carbon without rotation in a Cressington
CFE-60 (Cressington, Watford, UK). Platelets were viewed at 100 kV in a
JEOL 1200-EX electron microscope (Hartwig et al., 1996; Kovacsovics and
Hartwig, 1996)

In Vitro Phagocytic Assay

[0180] Monocytic THP-1 cells were cultured for 7 days in RPMI 1640 cell
culture media supplemented with 10% fetal bovine serum, 25 mM Hepes, 2 mM
glutamine and differentiated using 1 ng/ml TGFP and 50 nM 1,25-(OH)2
vitamin D3 for 24 hours, which is accompanied by increased expression of
CR3 (Simon et al., 2000). CR3 expression was monitored by flow cytometry
using a PE-conjugated anti-human CD11b/Mac-1 mAb. Undifferentiated or
differentiated THP-1 cells (2×106/ml) were plated onto 24-well
plates and allowed to adhere for 45 minutes at 37° C. The adherent
undifferentiated or differentiated macrophages were activated by the
addition of 15 ng/ml PMA for 15 min. CM-range-labeled, chilled or room
temperature platelets (107/well), previously subjected to different
treatments were added to the undifferentiated or differentiated
phagocytes in Ca2+- and Mg2+-containing HBSS and incubated for
30 min at 37° C. Following the incubation period, the phagocyte
monolayer was washed with HBSS for 3 times, and adherent platelets were
removed by treatment with 0.05% trypsin/0.53 mM EDTA in HBSS at
37° C. for 5 min followed by 5 mM EDTA at 4° C. to detach
the macrophages for flow cytometric analysis of adhesion or ingestion of
platelets (Brown et al., 2000). Human CM-Orange-labeled, chilled or room
temperature platelets all expressed the same amount of the platelet
specific marker CD61 as freshly isolated unlabeled platelets (not shown).
CM-Orange-labeled platelets incubated with macrophages were resolved from
the phagocytes according to their forward and side scatter properties.
The macrophages were gated, 10,000 events acquired for each sample, and
data analyzed with CELLQuest software (Becton Dickenson).
CM-Orange-labeled platelets that associate with the phagocyte population
have a shift in orange fluorescence (FIG. 6A and FIG. 6B, ingested, y
axis). These platelets were ingested rather than merely adherent, because
they failed to dual label with the FITC-conjugated mAb to CD61.

Immunolabeling and Flow Cytometry of Platelets

[0181] Washed murine or human platelets (2×106) were analyzed
for surface expression of CD62P, CD61, or surface bound IgM and IgG after
chilling or room temperature storage by staining with
fluorophore-conjugated Abs (5 μg/ml) for 10 min at 37° C.
Phosphatidylserine exposure by chilled or room temperature platelets was
determined by resuspending 5 μl of platelets in 400 μl of HBSS
containing 10 mM Ca2+ with 10 μg/ml of FITC-conjugated annexin-V.
As a positive control for PS exposure, platelet suspensions were
stimulated with 1 μM A23187. Fibrinogen binding was determined by the
addition of Oregon Green-fibrinogen for 20 min at room temperature. All
platelet samples were analyzed immediately by flow cytometry. Platelets
were gated by forward and side scatter characteristics.

Intravital Microscopy Experiments

[0182] Animal preparation, technical and experimental aspects of the
intravital video microscopy setup have been described (von Andrian,
1996). Six to eight week-old mice of both sexes were anesthetized by
intraperitoneal injection of a mixture of Xylazine and Ketamin. The right
jugular vein was catheterized with PE-10 polyethylene tubing. The lower
surface of the left liver lobe was surgically prepared and covered by a
glass cover slip for further in vivo microscopy as described (McCuskey,
1986). 108 chilled platelets and room temperature platelets labeled
with CMFDA and TRITC respectively were mixed 1:1 and administered
intravenously. The circulation of labeled platelets in liver sinusoids
was followed by video triggered stroboscopic epi-illumination. Ten video
scenes were recorded from 3 centrilobular zones at each indicated time
point. The ratio of cooled (CMFDA)/RT (TRITC) adherent platelets in the
identical visualized field was calculated. Confocal microscopy was
performed using a Radiance 2000 MP confocal-multiphoton imaging system
connected to an Olympus BX 50 WJ upright microscope (Biorad, Hercules,
Calif.), using a 10× water immersion objective. Images were
captured and analyzed with Laser Sharp 2000 software (Biorad) (von
Andrian, 2002).

Platelet Aggregation in Shed Blood

[0183] We used a flow cytometric method to analyze aggregate formation by
platelets in whole blood emerging from a wound as described for primates
(Michelson et al., 1994). We injected 108 CMFDA labeled room
temperature murine platelets into syngeneic wild type mice and 108
CMFDA labeled, chilled platelets into CR3-deficient mice. Twenty-four
hours after the platelet infusion, a standard bleeding time assay was
performed, severing a 3-mm segment of a mouse tail (Denis et al., 1998).
The amputated tail was immersed in 100 μl 0.9% isotonic saline at
37° C. The emerging blood was collected for 2 min., and 0.1 volume
of Aster-Jandl anticoagulant added and followed immediately with 1%
paraformaldehyde (final concentration). Peripheral blood was obtained by
retroorbital eye plexus bleeding in parallel as described above and
immediately fixed with 1% paraformaldehyde (final concentration). To
analyze the number of aggregates in vivo by flow cytometry, the shed
blood emerging from the bleeding time wound, as well as a peripheral
whole blood sample, were diluted and labeled with PE-conjugated
anti-murine GP1bα mAb pOp4 (5 μg/ml, 10 min.). Platelets were
discriminated from red cells and white cells by gating according to their
forward scatter characteristics and GP1bα positivity. A histogram
of log forward light scatter (reflecting platelet size) versus
GP1bα binding was then generated. In the peripheral whole blood
samples, analysis regions were plotted around the GP1bα-positive
particles to include 95% of the population on the forward scatter axis
(region 1) and the 5% of particles appearing above this forward light
scatter threshold (region 2). Identical regions were used for the shed
blood samples. The number of platelet aggregates in shed blood as a
percentage of the number of single platelets was calculated from the
following formula: [(number of particles in region 2 of shed
blood)-(number of particles in region 2 of peripheral blood)]/(number of
particles in region 1 of shed blood)×100%. The infused platelets
were identified by their CMFDA labeling and discriminated from the CMFDA
negative non-infused platelets.

[0184] Room temperature CM-Orange-labeled room temperature platelets
(108) were injected into wild type mice and CM-Orange-chilled
labeled platelets (108) into CR3 deficient mice. Twenty-four hours
after platelet infusion the mice were bled and the platelets isolated.
Resting or thrombin activated (1 U/ml, 5 min) platelet suspensions
(2×108) were diluted in PBS and either stained with
FITC-conjugated anti-mouse P-selectin mAb or with 50 μg/ml Oregon
Green-conjugated fibrinogen for 20 min at room temperature. Platelet
samples were analyzed immediately by flow cytometry. Transfused and
non-transfused platelets were gated by their forward scatter and
CM-Orange fluorescence characteristics. P-selectin expression and
fibrinogen binding were measured for each CM-Orange positive and negative
population before and after stimulation with thrombin.

Statistics

[0185] The intravital microscopy data are expressed as means±SEM.
Groups were compared using the nonpaired t test. P values <0.05 were
considered significant. All other data are presented as the mean±SD.

Results

The Clearance of Chilled Platelets Occurs Predominantly in the Liver and
is Independent of Platelet Shape.

[0186] Mouse platelets kept at room temperature (RT) and infused into
syngeneic mice disappear at fairly constant rate over time for about 80
hours (FIG. 1A). In contrast, approximately two-thirds of mouse platelets
chilled at ice-bath temperature and rewarmed (Cold) before injection
rapidly disappear from the circulation as observed previously in humans
and mice (Becker et al., 1973; Berger et al., 1998). Chilled and rewarmed
platelets treated with the cell-permeable calcium chelator EGTA-AM and
the actin filament barbed end capping agent cytochalasin B
(Cold+CytoB/EGTA) to preserve their discoid shape (Winokur and Hartwig,
1995), left the circulation as rapidly as chilled, untreated platelets
despite the fact that these platelets were fully functional as determined
by thrombin-, ADP- or collagen related peptide- (CRP) induced aggregation
in vitro (FIG. 1B). The recoveries of infused platelets immediately
following transfusion were 50-70%, and the kinetics of platelet
disappearance were indistinguishable whether we used 111Indium or
CMFDA to label platelets. The relative survival rates of room temperature
and chilled mouse platelets resemble the values reported previously for
identically treated mouse (Berger et al., 1998) and human platelets
(Becker et al., 1973).

[0187] FIG. 1C shows that the organ destinations of room temperature and
chilled mouse platelets differ. Whereas room-temperature platelets
primarily end up in the spleen, the liver is the major residence of
chilled platelets removed from the circulation. A greater fraction of
radionuclide detected in the kidneys of animals receiving
111Indium-labeled chilled compared with room-temperature platelets
at 24 hours may reflect a more rapid degradation of chilled platelets and
delivery of free radionuclide to the urinary system. One hour after
injection the organ distribution of platelets labeled with CMFDA was
comparable to that of platelets labeled with 111Indium. In both
cases, 60-90% of the labeled chilled platelet population deposited in the
liver, ˜20% in the spleen and ˜15% in the lung. In contrast,
a quarter of the infused room temperature platelets distributed equally
among the liver, spleen and lung.

Chilled Platelets Co-Localize with Liver Macrophages (Kupffer Cells).

[0188] The clearance of chilled platelets by the liver and the evidence
for platelet degradation is consistent with recognition and ingestion of
chilled platelets by Kupffer cells, the major phagocytic scavenger cells
of the liver. FIG. 1D shows the location of phagocytotic Kupffer cells
and adherent chilled CMFDA-labeled platelets in a representative confocal
micrograph of a mouse liver section 1 hour after transfusion. Sinusoidal
macrophages were visualized by the injection of 1 μM carboxyl modified
polystyrene microspheres marked with Nile-red. Co-localization of
transfused platelets and macrophages is indicated in yellow in the merged
micrograph of both fluorescence emissions. The chilled platelets localize
with Nile-red-labeled cells preferentially in the periportal and midzonal
domains of liver acini, sites rich in sinusoidal macrophages
(Bioulac-Sage et al., 1996; MacPhee et al., 1992).

CR3-Deficient Mice do not Rapidly Clear Chilled Platelets.

[0189] CR3 (αMβ2 integrin; CD11b/CD18; Mac-1) is a
major mediator of antibody independent clearance by hepatic macrophages.
FIG. 2a shows that chilled platelets circulate in CR3-deficient animals
with the same kinetics as room-temperature platelets, although the
clearance of both platelet populations is shorter in the CR3-deficient
mouse compared to that in wild-type mice (FIG. 1A). The reason for the
slightly faster platelet removal rate by CR3-deficient mice compared to
wild-type mice is unclear. Chilled and rewarmed platelets also clear
rapidly from complement factor 3 C3-deficient mice (FIG. 2c), missing a
major opsonin that promotes phagocytosis and clearance via CR3 and from
von Willebrand factor (vWf) deficient mice (Denis et al., 1998) (FIG.
2b).

Chilled Platelets Adhere Tightly to Kupffer Cells In Vivo.

[0190] Platelet adhesion to wild-type liver sinusoids was further
investigated by intravital microscopy, and the ratio between chilled and
room temperature stored adherent platelets infused together was
determined. FIG. 3 shows that both chilled and room temperature platelets
attach to sinusoidal regions with high Kupffer cell density (FIGS. 3a and
3b), but that 2.5 to 4-times more chilled platelets attach to Kupffer
cells in the wild-type mouse than room-temperature platelets (FIG. 3c).
In contrast, the number of platelets adhering to Kupffer cells in
CR3-deficient mice was independent of chilling or room temperature
exposure (FIG. 3c).

[0191] Because GP1bα, a component of the GP1b-IX-V receptor complex
for vWf, can bind CR3 under certain conditions in vitro (Simon et al.,
2000), we investigated GP1bα as a possible counter receptor on
chilled platelets for CR3. The O-sialoglycoprotein endopeptidase cleaves
the 45-kDa N-terminal extracellular domain of the murine platelet
GP1bα, leaving other platelet receptors such as
(αIIbβ3, α2α1,
GPVI/FcRγ-chain and the protease-activated receptors intact
(Bergmeier et al., 2001). Hence, we stripped this portion of the
extracellular domain of GP1bα from mouse platelets with
O-sialoglycoprotein endopeptidase (FIG. 4A inset) and examined their
survival in mice following room temperature or cold incubation. FIG. 4A
shows that chilled platelets no longer exhibit rapid clearance after
cleavage of GP1bα. In addition, GP1bα depleted room
temperature-treated platelets have slightly elongated survival times
(˜5-10%) when compared to the GP1bα-containing
room-temperature controls.

Chilling does not Affect Binding of Activated vWf to the Platelet
vWf-Receptor but Induces Clustering of GP1bα on the Platelet
Surface.

[0192] FIG. 4B shows that botrocetin-activated vWf binds GP1bα
equally well on room temperature as on cold platelets, although chilling
of platelets leads to changes in the distribution of GP1bα on the
murine platelet surface. GP1bα molecules, identified by immunogold
labeled monoclonal murine anti-GP1bα antibodies, form linear
aggregates on the smooth surface of resting discoid platelets at room
temperature (FIG. 4C, RT). This arrangement is consistent with
information about the architecture of the resting blood platelet. The
cytoplasmic domain of GP1bα binds long filaments curving with the
plane of the platelet membrane through the intermediacy of filamin A
molecules (Hartwig and DeSisto, 1991). After chilling (FIG. 4C, Chilled)
many GP1bα molecules organize as clusters over the platelet
membrane deformed by internal actin rearrangements (Hoffmeister et al.,
2001; Winokur and Hartwig, 1995).

Recognition of Platelet GP1bα by CR3-Mediates Phagocytosis of
Chilled Human Platelets In Vitro.

[0193] Differentiation of human monocytoid THP-1 cells using TGF-β1
and 1,25-(OH)2 Vitamin D3 increases expression of CR3 by
˜2-fold (Simon et al., 1996). Chilling resulted in 3-fold increase
of platelet phagocytosis by undifferentiated THP-1 cells and a
˜5-fold increase by differentiated THP-1 cells (FIGS. 5B and 5c),
consistent with mediation of platelet uptake by CR3. In contrast, the
differentiation of THP-1 cells had no significant effect on the uptake of
room temperature stored platelets (FIGS. 5A and 5c). To determine if
GP1bα is the counter receptor for CR3-mediated phagocytosis on
chilled human platelets, we used the snake venom metalloprotease
mocarhagin, to remove the extracellular domain of GP1bα (Ward et
al., 1996). Removal of human GP1bα from the surface of human
platelets with mocarhagin reduced their phagocytosis after chilling by
˜98% (FIG. 5C).

Exclusion of Other Mediators of Cold-Induced Platelet Clearance

[0194] Table 1 shows results of experiments that examined whether cooling
affected the expression of platelet receptors other than GP1bα or
their interaction with ligands. These experiments revealed no detectable
effects on the expression of P-selectin,
αIIbβ3-integrin density or on
αIIbβ3 fibrinogen binding, a marker of
αIIbβ3 activation. Chilling also did not increase
phosphatidylserine (PS) exposure, an indicator of apoptosis, nor did it
change platelet binding of IgG or IgM immunoglobulins.

[0195] The binding of fluorescently labeled antibodies or ligands against
various receptors on chilled-rewarmed or room temperature human and
murine platelets was measured by flow cytometry. The data are expressed
as the ratio between the mean fluorophore bound to the surface of chilled
versus room temperature platelets (mean±SD, n=3-4).

[0196] Despite their rapid clearance in wild type mice, CM-Orange or CMFDA
labeled chilled platelets were functional 24 h after infusion into
CR3-deficient mice, as determined by three independent methods. First,
chilled platelets incorporate into platelet aggregates in shed blood
emerging from a standardized tail vein bleeding wound (FIG. 6).
CMFDA-positive room temperature platelets transfused into wild type mice
(FIG. 6B) and CNIFDA-positive chilled platelets transfused into
CR3-deficient mice (FIG. 6d) formed aggregates in shed blood to the same
extent as CMFDA-negative platelets of the recipient mouse. Second, as
determined by platelet surface exposure of the fibrinogen-binding site on
αIIbβ3 24 hours after transfusion of
CM-Orange-labeled chilled and rewarmed platelets into CR3 deficient mice
following ex vivo stimulation by thrombin. Third, CM-Orange platelets
chilled and rewarmed were fully capable of upregulation of P-selectin in
response to thrombin activation (FIG. 6e).

Discussion

[0197] Cold-Induced Platelet Shape Change Alone does not Lead to Platelet
Clearance In Vivo

[0198] Cooling rapidly induces extensive platelet shape changes mediated
by intracellular cytoskeletal rearrangements (Hoffmeister et al., 2001;
White and Krivit, 1967; Winokur and Hartwig, 1995). These alterations are
partially but not completely reversible by rewarming, and rewarmed
platelets are more spherical than discoid. The idea that preservation of
platelet discoid shape is a major requirement for platelet survival has
been a dogma, despite evidence that transfused murine and baboon
platelets activated ex vivo by thrombin circulate normally with extensive
shape changes (Berger et al., 1998; Michelson et al, 1996). Here we have
shown that chilling leads to specific changes in the platelet surface
that mediate their removal independently of shape change, and that the
shape change per se does not lead to rapid platelet clearance. Chilled
and rewarmed platelets, preserved as discs with pharmacological agents,
clear with the same speed as untreated chilled platelets, and misshapen
chilled and rewarmed platelets circulate like room temperature maintained
platelets in CR3-deficient mice. The small size of platelets may allow
them to remain in the circulation, escaping entrapment despite these
extensive shape deformities.

Receptors Mediating Clearance of Chilled Platelets: CR3 and GP1bα

[0199] The normal platelet life span in humans is approximately 7 days
(Aas, 1958; Ware et 2000). The incorporation of platelets into small
blood clots engendered by continuous mechanical stresses undoubtedly
contributes to platelet clearance, because massive clotting reactions,
such as occur during disseminated intravascular coagulation, cause
thrombocytopenia (Seligsohn, 1995). The fate of platelets in such
clotting reactions differs from that of infused ex vivo-activated
platelets such as in the experiments of Michelson et al (Michelson et
al., 1996) and Berger et al (Berger et al., 1998), because in vivo
platelet stimulation occurs on injured vessel walls, and the activated
platelets rapidly sequester at these sites.

[0200] Isoantibodies and autoantibodies accelerate the phagocytic removal
of platelets by Fc-receptor-bearing macrophages in individuals sensitized
by immunologically incompatible platelets or in patients with autoimmune
thrombocytopenia, but otherwise little information exists regarding
mechanisms of platelet clearance. We showed, however, that the quantities
of IgG or IgM bound to chilled or room-temperature human platelets are
identical, implying that binding of platelet-associated antibodies to
Fc-receptors does not mediate the clearance of cooled platelets. We also
demonstrated that chilling of platelets does not induce detectable
phosphatidylserine (PS) exposure on the platelet surface in vitro
militating against PS exposure and the involvement of scavenger receptors
in the clearance of chilled platelets.

[0201] Although many publications have referred to effects of cold on
platelets as "activation", aside from cytoskeletally-mediated shape
changes, chilled platelets do not resemble platelets activated by stimuli
such as thrombin or ADP. Normal activation markedly increases surface
P-selectin expression, a consequence of secretion from intracellular
granules (Berman et al., 1986). Chilling of platelets does not lead to
up-regulation of P-selectin (Table 1), but the clearance of chilled
platelets isolated from wild-type or P-selectin-deficient mice is equally
rapid (Berger et al., 1998). Activation also increases the amount of
αIIbβ3-integrin and its avidity for fibrinogen
(Shattil, 1999), but cooling does not have these effects (Table 1). The
normal survival of thrombin-activated platelets is consistent with our
findings.

[0202] We have shown that CR3 on liver macrophages is primarily
responsible for the recognition and clearance of cooled platelets. The
predominant role of CR3 bearing macrophages in the liver in clearance of
chilled platelets despite abundant CR3-expressing macrophages in the
spleen is consistent with the principally hepatic clearance of IgM-coated
erythrocytes (Yan et al., 2000) and may reflect blood filtration
properties of the liver that favor binding and ingestion by macrophage
CR3. The extracellular domain of GP1bα binds avidly to CR3, and
under shear stress in vitro supports the rolling and firm adhesion of
THP-1 cells (Simon et al., 2000). Cleavage of the extracellular domain of
murine GP1bα results in normal survival of chilled platelets
transfused into mice. GP1bα depletion of human chilled platelets
greatly reduces phagocytosis of the treated platelets by macrophage-like
cells in vitro. We propose, therefore, that GP1bα is the
co-receptor for liver macrophage CR3 on chilled platelets leading to
platelet clearance by phagocytosis.

[0203] The normal clearance of cold platelets lacking the N-terminal
portion of GP1bα rules out the many other CR3-binding partners,
including molecules expressed on platelet surfaces as candidates for
mediating chilled platelet clearance. These ligand candidates include
ICAM-2, fibrinogen bound to the platelet integrin
αIIbβ3, iC3b, P-selectin, glucosaminoglycans, and
high molecular weight kininogen. We excluded deposition of the opsonic
C3b fragment iC3b as a mechanism for chilled platelet clearance using
mice deficient in complement factor 3, and the expression level of
αIIbβ3 and fibrinogen binding are also unchanged
after chilling of platelets.

Binding to Activated vWf and Cold-Induced Binding to CR3 Appear to be
Separate Functions of GP1bα.

[0204] GP1bα on the surface of the resting discoid platelet exists
in linear arrays (FIG. 5) in a complex with GP1bα, GP1X and V,
attached to the submembrane actin cytoskeleton by filamin-A and Filamin B
(Stossel et al., 2001). Its role in hemostasis is to bind the activated
form of vWf at sites of vascular injury. GP1bα, binding to
activated vWf is constitutive and requires no active contribution from
the platelet, since activated vWf binds equally well to GP1bα on
resting or on stimulated platelets. Stimulation of platelets in
suspension by thrombin and other agonists causes GP1bα to
redistribute in part from the platelet surface into an internal membrane
network, the open canalicular system, but does not lead to platelet
clearance in vivo (Berger et al., 1998; Michelson et al., 1996) or to
phagocytosis in vitro (unpublished observations). Cooling of platelets
however, causes GP1bα clustering rather than internalization. This
clustering is independent of barbed end actin assembly, because it occurs
in the presence of cytochalasin B.

[0205] Despite cold's promoting recognition of platelet GP1bα by
CR3, it has no effect on interaction between GP1bα and activated
vWf in vitro, and chilled platelets transfused into vWf-deficient mice
disappear as rapidly as in wild-type mice. The separability of
GP1bα's interaction with vWf and CR3 suggests that selective
modification of GP1bα. might inhibit cold-induced platelet
clearance without impairment of GP1bα's hemostatically important
reactivity with vWf. Since all tests of platelet function of cooled
platelets in vitro and after infusion into CR3-deficient mice yielded
normal results, suitably modified platelets would predictably be
hemostatically effective.

Physiological Importance of Cold-Induced Platelet Clearance.

[0206] Although gross platelet shape changes become obvious only at
temperatures below 15° C., accurate biochemical analyses show that
cytoskeletal alterations and increased responsiveness to thrombin are
detectable as the temperature falls below 37° C. (Faraday and
Rosenfeld, 1998; Hoffmeister et al., 2001; Tablin et al., 1996). We refer
to those changes as "priming" because of the many functional differences
that remain between cold-exposed and thrombin- or ADP-stimulated
platelets. Since platelet activation is potentially lethal in coronary
and cerebral blood vessels subjected to core body temperatures, we have
proposed that platelets are thermosensors, designed to be relatively
inactive at the core body temperature of the central circulation but to
become primed for activation at the lower temperatures of external body
surfaces, sites most susceptible to bleeding throughout evolutionary
history (Hoffmeister et al., 2001). The findings reported here suggest
that irreversible changes in GP1bα are the reason for the clearance
of cooled platelets. Rather than allowing chilled platelets to circulate,
the organism clears low temperature-primed platelets by phagocytosis.

[0207] A system involving at least two clearance pathways, one for removal
of locally activated platelets and another for taking out excessively
primed platelets (FIG. 7), can possibly explain why chilled platelets
circulate and function normally in CR3-deficient mice and have a slightly
prolonged circulation following removal of GP1bα. We propose that
some primed platelets enter microvascular clots on a stochastic basis.
Others are susceptible to repeated exposure to body surface temperature,
and this repetitive priming eventually renders these platelets
recognizable by CR3-bearing liver macrophages. Platelets primed by
chilling are capable of normal hemostatic function in CR3-deficient mice,
and coagulation contributes to their clearance. However, the slightly
shorter survival time of autologous platelets in CR3-deficient mice
examined is probably not ascribable to increased clearance of normally
primed platelets in microvascular clots, because the clearance rate of
refrigerated platelets was indistinguishable from that of platelets kept
at room temperature.

[0253] αMβ2 (CR3) has a cation-independent
sugar-binding lectin site, located "C-T" to its I-domain (Thornton et al,
J. Immonol. 156, 1235-1246, 1996), which binds to mannans, glucans and
N-Acetyl-D-glucosamine (GlcNAc). Since CD16b/αMβ2
membrane complexes are disrupted by β-glucan,
N-Acetyl-D-galactosamine (GalNAc), and methyl-α-mannoside, but not
by other sugars, it is believed that this interaction occurs at the
lectin site of the αMβ2 integrin (CR3) (Petty et al,
J. Leukoc. Biol. 54, 492-494, 1993; Sehgal et al, J. Immunol. 150,
4571-4580, 1993).

[0254] The lectin site of αMβ2 integrin has a broad
sugar specificity (Ross, R. Critical Reviews in Immunology 20, 197-222,
2000). Although sugar binding to lectins is usually of low affinity,
clustering can cause a more robust interaction by increasing avidity. The
clustering of GP1bα following cooling, as shown by electron
microscopy, suggests such a mechanism. The most common hexosamines of
animal cells are D-glucosamine and D-galactosamine, mostly occurring in
structural carbohydrates as GlcNAc and GalNAc, suggesting that the
αMβ2 integrin lectin domain might also bind to
mammalian glycoproteins containing carbohydrates that are not covered by
sialic acid. The soluble form of GP1bα, glycocalicin, has a
carbohydrate content of 60% comprising N- as well as O-glycosidically
linked carbohydrate chains (Tsuji et al, J. Biol. Chem. 258, 6335-6339,
1983). Glycocalicin contains 4 potential N-glycosylation sites (Lopez, et
al, Proc. Natl. Acad. Sci., USA 84, 5615-5619, 1987). The 45 kDa region
contains two sites that are N-glycosylated (Titani et al, Proc Natl Acad
Sci 16, 5610-5614, 1987). In normal mammalian cells, four common core
structures of O-glycan can be synthesized. All of them may be elongated,
sialylated, fucosylated and sulfated to form functional carbohydrate
structures. The N-linked carbohydrate chains of GP1bα are of the
complex-type and di-, tri- and tetra-antennary structures (Tsuji et al,
J. Biol. Chem. 258, 6335-6339, 1983). They are sialylated GalNAc type
structures with an α(1-6)-linked fucose residue at the Asn-bound
GlcNAc unit. There is a structural similarity of Asn-linked sugar chains
with the Ser/Thr-linked: i.e., their position is of a common Gal-GlcNAc
sequence. Results suggested that removal of sialic acid and galactose has
no influence on the binding of vWf to glycocalicin, but partial removal
of GlcNac resulted in the inhibition of vWf binding (Korrel et al, FEBS
Lett 15, 321-326, 1988). A more recent study proposed that the
carbohydrate patterns are involved in maintaining an appropriate
functional conformation of the receptor, without participating directly
in the binding of vWf (Moshfegh et al, Biochem. Biophys. Res. Communic.
249, 903-909, 1998).

[0255] A role of sugars in the interaction between chilled platelets and
macrophages has the important consequence that covalent modification,
removal or masking of oligosaccharide residues could prevent this
interaction. We hypothesized that if such prevention does not impair
normal platelet function, we may be able to modify platelets and enable
cold platelet storage. Here, we show evidence that favor this hypothesis:
1) Saccharides inhibited phagocytosis of chilled platelets by macrophages
in vitro, and the specific sugars that are effective implicated
β-glucans as the relevant targets. Low concentrations of
β-GlcNAc were surprisingly effective inhibitors, consistent with the
idea that interference with a relatively small number of clustered sugars
may be sufficient to inhibit phagocytosis. Addition of sugars at
concentrations that maximally inhibited phagocytosis of chilled platelets
has no effect on normal GP1bα function (vWf-binding); 2) A
β-GlcNAc-specific lectin, but not other lectins, bound avidly to
chilled platelets; 3) Removal of GP1bα or β-GlcNAc residues
from platelet surfaces prevented this binding (since β-GlcNAc
removal exposed mannose residues, it did not prevent phagocytosis by
macrophages which have mannose receptors); 4) Blocking of exposed
β-Glucans on chilled platelets by enzymatic addition of galactose
markedly inhibited phagocytosis of chilled platelets by macrophages in
vitro and extended the circulation times of chilled platelets in normal
animals.

Effect of Monosaccharides on Phagocytosis of Chilled Platelets.

[0256] To analyze the effects of monosaccharides on platelet phagocytosis,
phagocytes (differentiated monocytic cell line THP-1) were incubated in
monosaccharide solutions at various concentrations, and the chilled or
room temperature platelets were added. Values in the Figures are
means±SD of 3-5 experiments comparing percentages of orange-positive
monocytes containing ingested platelets incubated with RT or chilled
platelets). While 100 mM D-glucose inhibited chilled platelet
phagocytosis by 65.5% (P<0.01), 100 mM D-galactose did not
significantly inhibit chilled platelet phagocytosis (n=3) (FIG. 8A). The
D-glucose α-anomer (α-glucoside) did not have an inhibitory
effect on chilled platelet phagocytosis, although 100 mM inhibited by
90.2% (FIG. 8B) In contrast, β-glucoside inhibited phagocytosis in a
dose-dependent manner (FIG. 8B). Incubation of the phagocytes with 100 mM
β-glucoside inhibited phagocytosis by 80% (p<0.05) and 200 mM by
97% (P<0.05), therefore we concluded that the β-anomer is
preferred. D-mannose and its α- and β-anomers
(methyl-α-D-mannopyranoside (FIG. 8C) and
methyl-β-D-mannopyranoside (FIG. 8C) had no inhibitory effect on
chilled or RT platelet phagocytosis. Incubation of phagocytes using 25 to
200 mM GlcNAc (N-acetyl-D-glucosamine) significantly inhibited chilled
platelet phagocytosis. Incubation with 25 mM GlcNac was sufficient to
inhibit the phagocytosis of chilled platelets by 86% (P<0.05) (FIG.
8D), whereas 10 μM of the β-anomer of GlcNAc inhibited the
phagocytosis of chilled platelets by 80% (p<0.01) (FIG. 8D). None of
the monosaccharides had an inhibitory effect on RT platelet phagocytosis.
Table 2 summarizes the inhibitory effects of monosaccharides at the
indicated concentrations on chilled platelet phagocytosis (**P<0.01,
*P<0.05). None of the monosaccharides inhibited thrombin or ristocetin
induced human platelet aggregation or induced α-granule secretion
as measured by P-selectin exposure.

Binding of Various Lectins to Room Temperature Platelets or Chilled
Platelets.

[0257] β-GlcNAc strongly inhibited chilled human platelet
phagocytosis in vitro at μM concentrations, indicating that GlcNac is
exposed after incubation of platelets in the cold. We then investigated
whether wheat germ agglutinin (WGA), a lectin with specificity towards
the terminal sugar (GlcNAc), binds more effectively to chilled platelets
than to room temperature platelets. Washed, chilled or room temperature
platelets were incubated with 2 μg/ml of FITC coupled WGA or FITC
coupled succinyl-WGA for 30 min at room temperature and analyzed by flow
cytometry. FIGS. 9A and 9B show the dot plots after incubation with
FITC-WGA of room temperature (RT) or chilled (Cold) human platelets. WGA
induces platelet aggregation and release of serotonin or ADP at
concentrations between 25-50 μg/ml WGA (Greenberg and Jamieson,
Biochem. Biophys. Acta 345, 231-242, 1974). Incubation with 2 μg/ml
WGA induced no significant aggregation of RT-platelets (FIG. 9A, RT
w/WGA), but incubation of chilled platelets with 2 μg/ml WGA induced
massive aggregation (FIG. 9B, Cold/w WGA). FIG. 9C shows the analysis of
FITC-WGA fluorescence binding to chilled or room temperature platelets.
To verify that the increase of fluorescence binding is not aggregation
related, we used succinyl-WGA (S-WGA), a dimeric derivate of the lectin
that does not induce platelet aggregation (Rendu and Lebret, Thromb Res
36, 447-456, 1984). FIGS. 9D and 9E show that succinyl-WGA (S-WGA) did
not induce aggregation of room temperature or chilled platelets, but
resulted the same increase in WGA binding to chilled platelets versus
room temperature platelets (FIG. 9F). The enhanced binding of S-WGA after
chilling of platelets cannot be reversed by warming of chilled platelets
to 37° C.

[0258] Exposed β-GlcNAc residues serve as substrate for a
β1,4galactosyltransferase enzyme that catalyses the linkage
Galβ-1GlcNAcβ1-R. In support of this prediction, masking of
β-GlcNAc residues by enzymatic galactosylation inhibited S-WGA
binding to cold platelets, phagocytosis of chilled platelets by THP-1
cells, and the rapid clearance of chilled platelets after transfusion
into mice. The enzymatic galactosylation, achieved with bovine
β1,4galactosyltransferase and its donor substrate UDP-Gal, decreased
S-WGA binding to chilled human platelets to levels equivalent to room
temperature platelets. Conversely, the binding of the galactose-specific
RCA I lectin increased by ˜2 fold after galactosylation.
UDP-Glucose and UDP alone had no effect on S-WGA or RCA I binding to
chilled or room temperature human platelets.

[0259] We found that the enzymatic galactosylation of human and mouse
platelets is efficient without addition of exogenous
β1,4galactosyltransferase. The addition alone of the donor substrate
UDP-Gal reduces S-WGA binding and increases RCA I binding to chilled
platelets, inhibits phagocytosis of chilled platelets by THP1 cells in
vitro, and prolongs the circulation of chilled platelets in mice. An
explanation for this unexpected finding is that platelets reportedly
slowly release endogenous galactosyltransferase activity. A least one
form of β1,4galactosyltransferases, β4Gal T1, is present in
human plasma, on washed human platelets and in the supernatant fluids of
washed platelets. Galactosyltransferases may associate specifically with
the platelet surface. Alternatively, the activity may be plasma-derived
and leak out of the platelet's open canalicular system. In either case,
modification of platelet glycans responsible for cold-mediated platelet
clearance is possible by simple addition of the sugar-nucleotide donor
substrate, UDP-Gal.

[0260] Importantly, both chilled and non-chilled platelets show the same
increase in RCA I binding after galactosylation, implying that
β-GlcNAc residues are exposed on the platelet surface independent of
temperature. However chilling is a requirement for recognition of
β-GlcNAc residues by S-WGA and by the αMβ2
integrin. We have demonstrated that chilling of platelets induces an
irreversible clustering of GP1b. Generally lectin binding is of low
affinity and multivalent interactions with high density of carbohydrate
ligands increases binding avidity. Possibly the local densities of
exposed β-GlcNAc on the surface of non-chilled platelets are too low
for recognition, but cold-induced clustering of GP1bα provides the
necessary density for binding to S-WGA or the αMβ2
integrin lectin domain. We confirmed by S-WGA and RCA-I binding flow
cytometry that UDP-Gal transfers galactose onto murine platelets in the
presence or absence of added galactosyl transferase and documented that
chilled, galactosylated murine platelets circulate and initially survive
significantly better than untreated room temperature platelets.

[0261] Although the earliest recoveries (<2 min) did not differ between
transfused RT, chilled and chilled, galactosylated platelets,
galactosylation abolished an initial platelet loss of about 20%
consistently observed with RT platelets.

[0262] Galactosylation of murine and human platelets did not impair their
functionality in vitro as measured by aggregation and P-selectin exposure
induced by collagen related peptide (CRP) or thrombin at concentrations
ranging from maximally effective to three orders of magnitude lower.
Importantly, the aggregation responses of unmodified and galactosylated
chilled human platelets to a range of ristocetin concentrations, a test
of the interaction between GP1b and activated VWF, were indistinguishable
or slightly better. The attachment points for N-linked glycans on
GP1bα are outside of the binding pocket for VWF. Moreover, mutant
GP1bα molecules lacking N-linked glycans bind VFW tightly.

[0264] We localized the exposed β-GlcNAc residues mediating
αMβ2 lectin domain recognition of GP1bα
N-glycans. The extracellular domain of GP1bα contains 60% of total
platelet carbohydrate content in the form of N- and O-glycosidically
linked carbohydrate chain. Accordingly, binding of peroxidase-labeled WGA
to GP1bα is easily detectable in displays of total platelet
proteins resolved by SDS-PAGE, demonstrating that GP1bα contains
the bulk of the β-GlcNAc-residues on platelets, and binding of WGA
to GP1bα is observable in GP1bα immunoprecipitates. UDP-Gal
with or without added galactosyltransferase diminishes S-WGA binding to
GP1bα, whereas RCA I binding to GP1bα increases. These
findings indicate that galactosylation specifically covers exposed
β-GlcNAc residues on GP1bα. Removal of the N-terminal 282
residues of GP1bα from human platelet surfaces using the snake
venom protease mocarhagin, which inhibited phagocytosis of human
platelets by THP-1 cells in vitro, reduces S-WGA binding to chilled
platelets nearly equivalent to S-WGA room temperature binding levels. WGA
binds predominantly to the N-terminus of GP1bα released by
mocarhagin into quadratureplatelet supernatant fluids as a polypeptide
band of 45 kDa recognizable by the monoclonal antibody SZ2 specific for
that domain. The glycans of this domain are N-linked. A small portion of
GP1bα remains intact after mocarhagin treatment, possibly because
the open canalicular system of the platelet sequesters it.
Peroxidase-conjugated WGA weakly recognizes the residual platelet
associated GP1bα C-terminus after mocarhagin cleavage, identifiable
with monoclonal antibody WM23.

[0265] The cold-induced increase in binding of human platelets to
αMβ2 integrin and to S-WGA occurs rapidly (within
minutes). The enhanced binding of S-WGA to chilled platelets remained
stable for up to 12 days of refrigerated storage in autologous plasma.
RCA I binding remained equivalent to room temperature levels under the
same conditions. Galactosylation doubled the binding of RCA I lectin to
platelets and reduced S-WGA binding to baseline RT levels. The increase
in RCA I and decrease in S-WGA binding were identical whether
galactosylation proceeded or followed storage of the platelets in
autologous plasma for up to 12 days. These findings indicate that
galactosylation of platelets to inhibit lectin binding is possible before
or after refrigeration and that the glycan modification is stable during
storage for up to 12 days. Platelets stored at room temperature rapidly
lose responsiveness to aggregating agents; this loss does not occur with
refrigeration. Accordingly, refrigerated platelets with or without
galactosylation, before or after storage, retained aggregation
responsiveness to thrombin for up to 12 days of cold storage.

[0266] The enzyme β-hexosaminidase catalyzes the hydrolysis of
terminal β-D-N-acetylglucosamine (GlcNAc) and galactosamine (GalNAc)
residues from oligosaccharides. To analyze whether removal of GlcNAc
residues reduces the binding of WGA to the platelet surface, chilled and
room temperature washed human platelets were treated with 100 U/ml
β-Hex for 30 min at 37° C. FIG. 11A shows the summary of
FITC-WGA binding to the surface of room temperature or chilled platelets
obtained by flow cytometry before and after β-hexosaminidase
treatment. FITC-WGA binding to chilled platelets was reduced by 85% after
removal of GlcNac (n=3). We also checked whether, as expected, removal of
GP1bα from the platelet surface leads to reduced WGA-binding after
platelet chilling. GP1bα was removed from the platelet surface
using the snake venom mocarhagin (MOC), as described previously (Ward et
al, Biochemistry 28, 8326-8336, 1996). FIG. 11B shows that GP1bα
removal from the platelet surface reduced FITC-WGA binding to chilled
platelets by 75% and had little influence on WGA-binding to
GP1bα-depleted room temperature platelets (n=3). These results
indicate that WGA binds mostly to oligosaccharides on GP1bα after
chilling of human platelets, and it is very tempting to speculate that
the Mac-1 lectin site also recognizes these exposed sugars on GP1bα
leading to phagocytosis.

Masking of Human Platelet GlcNAc Residues by Galactose-Transfer Greatly
Reduces their Phagocytosis After Chilling In Vitro and Dramatically
Increases their Survival in Mice.

[0267] To achieve galactose transfer onto platelets, isolated human
platelets were incubated with 200 μM UDP-galactose and 15 mU/ml
galactose transferase for 30 min at 37° C., followed by chilling
or maintenance at room temperature for 2 h. Galactosylation reduced
FITC-WGA binding almost to resting room temperature levels. Platelets
were fed to the monocytes and platelet phagocytosis was analyzed as
described above. FIG. 12 shows that galactose transfer onto platelet
oligosaccharides reduces greatly chilled platelet (Cold) phagocytosis,
but does not affect the phagocytosis of room temperature (RT) platelets
(n=3). These results show that in vitro the phagocytosis of chilled
platelets can be reduced through coverage of exposed GlcNAc residues. We
tested whether this approach could be extended to animals and used to
increase the circulation time of chilled platelets. Murine platelets were
isolated and stained with CMFDA. Using the same approach of galactose
transfer described for human platelets above, wild type murine platelets
were galactosylated and chilled, or not, for 2 hours. 108 Platelets
were transfused into wild type mice and their survival determined. FIG.
13 shows the survival of these chilled, galactosylated murine platelets
relative to untreated platelets. Both platelets kept at room temperature
(RT) and the galactosylated chilled platelets (Cold+GalT) had almost
identical survival times, whereas chilled untreated platelets (Cold) were
cleared rapidly as expected. We believe galactosylated chilled platelets
will circulate in humans.

[0268] We noted that our control reaction, in which galactose transferase
was heat-inactivated also resulted in glycan modification of platelets as
occurred in the experimental reaction with active galactose transferase,
as judged by WGA binding (FIG. 14A), in vitro phagocytosis results in
human platelets (FIG. 14B), and survival of murine platelets (FIG. 14C).
Therefore, we conclude that platelets contain galactose transferase
activity on their surface, which is capable of directing glycan
modification using only UDP-galactose without the addition of any
exogenous galactose transferase. Thus, glycan modification of platelets
can be achieved simply by incubation with UDP-galactose.

UDP-Galactose Incorporate into Human Platelets in a Time Dependent
Matter.

[0269] In another set of experiments we have shown that 14C-labeled
UDP-galactose incorporates into human platelets in a time dependent
manner in the presence or absence of the enzyme galactosyl transferase.
FIG. 15 shows the time course of 14C-labeled UDP-galactose
incorporation into washed human platelets. Human platelets were incubated
with 14C-labeled UDP-galactose for different time intervals in the
absence of galactosyl transferase. The platelets were then washed and the
14C radioactivity associated with platelets measured.

Example 3

Enzymatic Modification of Platelet β-Glycans Inhibit Phagocytosis of
Cooled Platelets by Macrophages In Vitro and Accommodate Normal
Circulation In Vivo

[0270] Our preliminary experiments have demonstrated the enzymatic
covering of GlcNAc residues on GP1bα using galactose-transfer
(glycan modification) onto chilled human platelet surfaces greatly
reduced their in vitro phagocytosis. One interpretation of these findings
is that GP1bα structure is altered on the surface of chilled human
and murine platelets. This causes the exposure or clustering of GlcNAc,
which is recognized by the lectin binding domain of αMβ2
leading to platelet removal. β-GlcNAc exposure can be measured by
WGA binding and possibly by binding of recombinant αMβ2 lectin
domain peptides. Resting human platelets bind WGA, which increases
greatly after chilling. We propose that galactose transfer (glycan
modification) will prevent GP1bα's interaction with
αMβ2-lectin but not with vWf. This modification (galactose
transfer onto platelet surface) leads to normal survival of chilled
platelets in WT mice as shown by our preliminary experiments.

Example 4

[0271] This example shows that the αMβ2 lectin site mimics WGA
and sugar modifications prevent the engagement of the recombinant lectin
site with chilled platelets. Dr. T. Springer (Corbi, et al., J. Biol.
Chem. 263, 12403-12411, 1988) provided the human αM cDNA and
several anti-αM antibodies. The smallest r-huαM construct
exhibiting lectin activity that has been reported includes its C-T and a
portion of its divalent cation binding region (residues 400-1098) (Xia et
al, J Immunol 162, 7285-7293, 1999). The construct is 6×His-tagged
for ease of purification. We first determined if the recombinant lectin
domain can be used as a competitive inhibitor of chilled platelet
ingestion in the phagocytic assay. Competition proved that the αM
lectin site mediates binding to the platelet surface and initiates
phagocytosis. As controls, a construct lacking the lectin-binding region
of αM was used and the recombinant protein was denatured. Lectin
binding domain functions as a specific inhibitor of chilled platelet
ingestion. We made a αM construct that include GFP and express and
labeled the αM-lectin binding site with FITC and used it to label
the surface of chilled platelets by flow cytometry. Platelets were
labeled with CMFDA. We found that chilled platelets bind more efficiently
to the αM lectin side of αMβ2 integrin compared to room
temperature platelets. The lectin side and whole αM-construct
(Mac-1) was expressed in Sf9 insect cells.

[0272] The platelet sugar chains are modified to inhibit the
platelet-oligosaccharide interaction with the r-huαM-lectin site.
The efficiency of sugar modifications is also monitored by inhibition of
the binding of fluorescent-lectin domain binding to platelets by flow
cytometry.

[0273] The recovery and circulation times of room temperature, chilled and
chilled-modified platelets are compared to establish that galactose
transfer onto chilled murine platelets results in longer circulating
platelets. Room temperature, chilled and chilled-modified platelets are
stained with CMFDA, and 108 platelets transfused into wild type mice
as described above. The mice are bled immediately (<2 min.), 30 min, 1
h, 2, 24, 48 and 72 hours after transfusion. The blood obtained is
analyzed using flow cytometry. The percentage of fluorescent labeled
platelets within the gated platelet population measured immediately after
injection is set as 100%. The recovery of fluorescently labeled platelets
obtained at the various time points is calculated accordingly.

Example 5

[0274] This example demonstrates that chilled, unmodified and chilled,
galactosylated (modified) platelets have hemostatic function in vitro and
in vivo. Chilled platelets are not "activated" in the sense of
agonist-stimulated platelets. Patients undergoing surgery under
hypothermic conditions may develop thrombocytopenia or show severe
hemostatic post-operative impairments. It is believed that under these
hypothermic conditions, platelets might lose their functionality.
However, when patients undergo hypothermic surgery, the whole organism is
exposed to hypothermia leading therefore to changes in multiple tissues.
Adhesion of non-chilled platelets to hepatic sinusoidal endothelial cells
is a major mechanism of cold preservation injury (Takeda, et al.
Transplantation 27, 820-828, 1999). Therefore, it is likely that it is
the interaction between cold hepatic endothelium and platelets, not
platelet chilling per se, that leads to deleterious consequences under
hypothermic conditions of surgery or trans-plantation of cold preserved
organs (Upadhya et al, Transplantation 73, 1764-1770, 2002). Two
approaches showed that chilled platelets have hemostatic function. In one
approach, the circulation of chilled platelets in
αMβ2-deficient mice facilitates studies of platelet function
after cooling. In the other approach, the function of modified chilled
and (presumably) circulating platelets was tested.

[0275] Human and murine unmodified and modified (galactosylated) chilled
platelets were tested for functionality, including in vitro aggregation
to agonists, P-selectin exposure and fibrinogen binding.

[0276] αMβ2 deficient or WT mice are transfused with murine
chilled/RT platelets modified or not, and allowed to circulate for 30
min., 2 and 24 hours. We determine if chilled platelets contribute to
clotting reactions caused by tail vein bleeding and if these platelets
bind agents such as fibrinogen after activation. We also determine how
chilled platelets, modified or not, contribute to clotting on ferric
chloride injured and exteriorized mouse mesenteries, an in vivo
thrombus-formation model that we developed. This method detects the
number of platelets adherent to injured vessels and has documented
impaired platelet vessel wall interactions of platelets lacking
glycoprotein V or β3-integrin function (Ni et al., Blood 98, 368-373
2001; Andre, et al. Nat Med 8, 247-252, 2002). Last, we determine the
storage parameters of the modified platelets.

[0277] In vitro platelet function is compared using aggregation with
thrombin and ADP and botrocetin induced vWf-binding to murine platelets.
Murine and human chilled platelets modified (galactosylated) or
unmodified platelets are normalized to a platelet concentration of
0.3×109/mm3, and aggregation induced using the various
agonists according to standard protocols (Bergmeier, et al. 2001 276,
25121-25126, 2001). To study vWf-binding we activate murine vWf using
botrocetin and analyze the binding of fluorescently labeled vWf to
chilled platelets modified or not in PRP (Bergmeier, et al. 2001 276,
25121-25126, 2001). To evaluate whether degranulation of platelets occurs
during modification, we also measure P-selectin exposure of chilled
murine and human platelets modified or not using fluorescent labeled
anti-P-selectin antibodies by flow cytometry (Michelson et al., Proc.
Natl. Acad. Sci., USA 93, 11877-11882, 1996).

[0278] 109 CMFDA-labeled platelets are transfused into mice, first
verifying that these platelets are functional in vitro. We determine
whether chilled platelets contribute to aggregation by transfusing
chilled or room temperature CMFDA-labeled platelets into αMβ2
deficient mice. At 30 min., 2 hours and twenty-four hours after the
infusion of platelets, a standard tail vein bleeding test is performed
(Denis, et al. Proc Natl Acad Sci USA 95, 9524-9529, 1998). The emerging
blood is fixed immediately in 1% formaldehyde and platelet aggregation is
determined by whole blood flow cytometry. Platelet aggregates appear as
bigger sized particles in the dot plot analysis. To verify that the
transfused platelets do not aggregate in the normal circulation we also
bleed the mice through the retroorbital eye plexus into an anticoagulant.
Platelets do not form aggregates under these bleeding conditions. The
emerging blood is fixed immediately and platelets are analyzed by flow
cytometry in whole blood as described above. Platelets are identified
through binding of a phycoerythrin-conjugated αIIbβ3
specific monoclonal antibody. The infused platelets in the blood sample
are identified by their CMFDA-fluorescence. Non-infused platelets are
identified by their lack of CMFDA fluorescence (Michelson, et al, Proc.
Natl. Acad. Sci., U.S.A. 93, 11877-11882, 1996). The same set of tests is
performed with CMFDA modified (galactosylated) chilled platelets
transfusing these platelets into αMβ2 and WT. This experiment
tests aggregation of chilled platelets modified or not in shed blood.

[0279] 109 CM-orange labeled unmodified chilled or room temperature
platelets are transfused into αMβ2 deficient mice to verify
that these platelets are functional in vitro. At 30 min., 2 h and
twenty-four hours after the infusion of CM-orange labeled platelets, PRP
is isolated as described and analyzed by flow cytometry. P-selectin
exposure is measured using an anti FITC-conjugated anti P-selectin
antibody (Berger, et al, Blood 92, 4446-4452, 1998). Non-infused
platelets are identified by their lack of CM-orange fluorescence. The
infused platelets in the blood sample are identified by their CM-orange
fluorescence. CM-orange and P-selectin positive platelets appear as
double positive fluorescently (CM-orange/FITC) stained platelets. To
verify that chilled platelets still expose P-selectin after thrombin
activation, PRP is activated through the addition of thrombin (1 U/ml, 2
min at 37° C.) and P-selectin exposure is measured as described.
To analyze the binding of fibrinogen to αIIbβ3,
isolated platelets are activated through the addition of thrombin (1
U/ml, 2 min, 37° C.) and Oregon-green coupled fibrinogen (20
μg/ml) added for 20 min at 37° C. (Heilmann, et al, Cytometry
17, 287-293, 1994). The samples are analyzed immediately by flow
cytometry. The infused platelets in the PRP sample are identified by
their CM-orange fluorescence. CM-orange and Oregon-green positive
platelets appear as double positive fluorescently stained
(CM-orange/Oregon green) platelets. The same sets of experiments are
performed with CM-orange labeled modified (galactosylated) chilled
platelets transfused into αMβ2 deficient and WT mice.

Example 6

In Vivo Thrombosis Model

[0280] First, we show the delivery of RT and unmodified chilled platelets
to injured endothelium of αMβ2 deficient mice using double
fluorescently labeled platelets. The resting blood vessel is monitored
for 4 min., then ferric chloride (30 μl of a 250-mM solution) (Sigma,
St Louis, Mo.) is applied on top of the arteriole by superfusion, and
video recording resumed for another 10 min. Centerline erythrocyte
velocity (Vrbc) is measured before filming and 10 min after ferric
chloride injury. The shear rate is calculated on the basis of
Poiseuille's law for a Newtonian fluid (Denis, et al, Proc Natl Acad Sci
U S A 95, 9524-9529, 1998). These experiments show if chilled platelets
have normal hemostatic function. We repeat these experiments in WT mice
comparing RT and galactosylated chilled platelets using two different,
fluorescently labeled platelet populations injected into the same mouse
and analyze the thrombus formation and incorporation of both platelet
populations.

[0281] We then compare in vitro platelet functions and survival and in
vivo hemostatic activity of chilled and modified chilled murine platelets
stored for 1, 5, 7 and 14 days under refrigeration as described above. We
compare the recovery and circulation times of these stored chilled and
modified chilled platelets and prove that: 1) the modification through
galactose transfer onto chilled murine platelets is stable after the long
term refrigeration; and 2) that these platelets function normally.
Survival experiments are performed as described above. We use WGA
binding, to verify that GlcNAc residues remain covered by galactose after
the longer storage time points. As an ultimate test that these modified,
stored platelets are functionally intact and contribute to hemostasis, we
transfuse them into total-body-irradiated mice (Hoyer, et al, Oncology
49, 166-172, 1992). To obtain the sufficient numbers of platelets, we
inject mice with commercially available murine thrombopoietin for seven
days to increase their platelet count (Lok, et al. Nature 369, 565-558,
1994). Isolated platelets are modified using the optimized galactose
transfer protocol, stored under refrigeration, transfused, and tail vein
bleeding times measured. Since unmodified chilled platelets do not
persist in the circulation, a comparison of modified cooled platelets
with room temperature stored platelets is not necessary at this point.
The murine platelets are stored under refrigeration in standard test
tubes. If a comparison with room temperature stored murine platelets is
necessary we switch to primate platelets. Rather than engineer special
down-scale, gas-permeable storage containers to accommodate mouse
platelets, such comparisons are more appropriate for primates (including
humans) for which room temperature storage bags have been designed.

[0283] It should be understood that the preceding is merely a detailed
description of certain preferred embodiments. It therefore should be
apparent to those skilled in the art that various modifications and
equivalents can be made without departing from the spirit and scope of
the invention. It is intended to encompass all such modifications within
the scope of the appended claims. All references, patents and patent
publications that are recited in this application are hereby incorporated
by reference herein in their entirety.

Example 8

Evaluation of the In Vivo Survival of UDP-Galactose Treated Platelets
Stored in the Cold

[0284] The technology for galactosylating human platelets with the use of
the activated carbohydrate substrate UDP-galactose may allow large-scale
human platelet storage under refrigeration (4° C.). Untreated
platelets stored at 4° C. are rapidly cleared from the
circulation. In contrast, untreated platelets stored at room temperature
survive for ˜5-7 days following transfusion. The present study is
intended to demonstrate that the galactosylated modified human platelets
circulate in vivo when infused autologously into individuals.

[0285] The reason for the removal of chilled platelets from the
circulation has recently been defined. Cooling of platelets causes
clustering of the platelet GPIb/V/IX complex on the platelet surface. The
αMβ2 integrin receptor (CR3, Mac-1) present on hepatic
macrophages recognizes clustered GPIbα molecules, and platelets are
ingested by the macrophages. The αMβ2 integrin receptor
contains a carbohydrate binding domain (lectin domain) that is critical
for the recognition of exposed β-N-acetylglucosamine (βGlcNAc)
residues on the platelet surface by macrophages. Covering of exposed
βGlcNAc residues by enzymatic galactosylation prevents recognition
and phagocytosis of chilled platelets. This has been extensively
demonstrated in a mouse model, where chilled and galactosylated murine
platelets have survival superior to that of room temperature stored
platelets. In vitro studies using human platelets indicate that
galactosylated platelets stored at 4° C. are likely also to
circulate when transfused into humans.

[0286] To determine and demonstrate that galactosylated modified human
platelets survive and circulate in vivo when infused autologously into
individuals. This will be determined by comparing the survival rates of
radiolabeled refrigerated (2°-8° C.) platelets with or
without galactosylation to radiolabeled non-galactosylated platelets
stored at room temperature (22°±2° C.) and in the cold
(Stored for 36 to 48 hrs).

[0287] The following describes a Phase I study in which in vivo recovery
and half-life of autologously-infused galactosylated platelets in normal,
healthy volunteer group subjects is determined.

[0288] Six (6) healthy donors will donate a unit of apheresis platelets.
The collected apheresis product will be divided into two bags. One bag
will have the platelets treated with UDP-galactose and stored under
refrigeration for 36-48 hours. The other platelet bag will either be
stored under refrigeration or as per current FDA guidelines at room
temperature for 36-48 hours. The two bags of platelets will each be
radiolabeled with a different radioactive isotope, 51Chromium or
111Indium and 5-10 mL of labeled platelets will be injected in the
healthy volunteers. Blood samples will be drawn before and at 2 hours
after the transfusion and then on days 1, 2, 3, 5, 7 and 10 after
reinfusion, and the post-transfusion recovery and survival of the
platelets will be determined.

[0289] The experimental material injected in the healthy volunteers will
be 5-10 mL aliquots of platelets that have been taken from the study
subjects, with or without modification by galactosylation and either
stored at room temperature (22±2° C.) or stored in the cold
(4±2° C.).

[0290] Upon confirmation of eligibility and enrollment in the study,
healthy donors will be recruited to donate a unit of platelets on the
Haemonetics MCS+ apheresis machine. This machine draws whole blood from a
donor's arm, centrifuges the blood to separate the platelets from the
plasma and the red cells, collects the platelets with a small amount of
plasma and returns most of the plasma and the red cells back to the
donor. The collected platelets and plasma will be divided into two bags.
Each bag will be weighed and the platelet count determined on the day of
collection, day 1 and day of infusion. After collection the platelets
will be rested for 1 hour. After the resting period one platelet bag will
be treated with a naturally occurring sugar substance, UDP-galactose.
This bag will be incubated for 1 hour at 37° C. and stored under
refrigeration. The other platelet bag will likewise be incubated for 1
hour at 37° C. and stored under refrigeration or as per current
FDA guidelines at room temperature. On Day 1 following collection a
sample from each bag will be sent to a microbiology lab for culture.

[0291] The platelet culture results will be recorded along with the
results of a gram stain sample that will be sent to the lab on the day of
reinfusion. If either report is positive the platelet units will not be
reinfused. The two bags of platelets will each be radiolabeled with a
different radioactive isotope, 51Chromium or 111Indium. Blood
samples will be drawn before and at 2 hours after and then on days 1, 2,
3, 5, 7 and 10 after the reinfusion. The blood samples will be analyzed
for radioactivity to determine the post-transfusion recovery and survival
of the platelets. Since the two units of platelets have been tagged with
different radioactive isotopes, we will be able to distinguish between
the platelets that were subjected to the UDP Galactose and those that are
untreated.

[0292] UDP-galactose (Uridine-5'-diphosphogalactose) is a natural sugar
compound found in the human body. It is used in this study as a donor for
the addition of galactose to the surface of the human platelets to be
transfused. The UDP-galactose was manufactured by Roche Diagnostics GmbH
and is over 97% pure. It contains trace quantities of by-products of the
manufacturing process. It was formulated and filled into syringes by a
licensed filling facility, and tested for sterility and pyrogenicity.

[0293] Blood samples taken from each study subject will be tested for
platelet count and anti-platelet antibodies before and at two weeks and
three months after the platelet infusion.

[0294] Between 5 and 10 mL of platelets radiolabeled with the two
different radioactive isotope, 51Chromium or 111Indium, will be
injected at day 0. Blood samples will be drawn before and at 2 hours and
on days 1, 2, 3, 5, 7 and 10 after reinfusion.

[0295] During each reinfusion, the subject will be carefully monitored for
adverse reactions, most usually fever, chills, dyspnea, urticaria or pain
(infusion site, chest pain or other), or significant changes in vital
signs. In addition, each subject will be queried during the follow up
period visits up to three months after the infusion to obtain information
on any occurrence of adverse events during that time. Non-modified and
modified platelets will be characterized by a number of in vitro analyses
including but not limited to: pH, pO2, pCO2, bicarbonate, hypotonic shock
response, morphology, extent of shape change, ATP levels, glucose, O2
consumption, p-Selectin, and Annexin V binding.